Antibody

ABSTRACT

Provided is an active ingredient of a pharmaceutical composition for treating myeloma. Specifically, provided is an antibody whose epitope is present in the region of the amino acid residue positions 20 to 109 of human integrin β 7 .

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/905,211, filed Jun. 18, 2020, which is a continuation of U.S.application Ser. No. 16/808,451, filed Mar. 4, 2020, which is acontinuation of U.S. application Ser. No. 15/751,574, filed on Feb. 9,2018 (now U.S. Pat. No. 10,654,931, issued May 19, 2020), which is aNational Stage of International Application No. PCT/JP2016/072688, filedon Aug. 2, 2016, which claims priority from Japanese Patent ApplicationNo. 2015-159240, filed on Aug. 11, 2015, the contents of all of whichare incorporated herein by reference in their entirety.

TECHNICAL FIELD

A novel antibody, a use thereof, and the like are disclosed.

BACKGROUND ART

Multiple myeloma, which is a typical example of a disease causingneoplastic growth of plasma cells, accounts for about 1% of all cancers,and accounts for a little more than 10% of all hematological malignanttumors. Multiple myeloma is a disease in which a plasma cell present inbone marrow becomes cancerous (becomes an abnormal plasma cell as aresult) and undergoes monoclonal growth.

In multiple myeloma, abnormal plasma cells (myeloma cells) spread to thebone marrow throughout the body and grow in every part of the bonemarrow throughout the entire body. When the abnormal plasma cells grow,various symptoms including bone breakage appear. The myeloma cellsproduce M protein, which is an abnormal immunoglobulin, to increase an Mprotein concentration in blood, and hence the blood becomes viscous.

The M protein does not function as an original antibody, whichrecognizes a foreign substance, such as a pathogen, which has enteredthe body. Accordingly, immunocompetence is reduced. Those phenomenaaffect many organs, and thus various symptoms occur. Typical symptomsare bone pain and damage, hypercalcaemia, nephropathy and renal failure,anemia, and the like.

At present, as treatment of multiple myeloma, proteasome inhibitors,iMIDs, such as thalidomide and a derivative thereof, specificallylenalidomide, and chemotherapy using, for example, melphalan incombination with prednisone, and hematopoietic stem cell transplantationare mainly employed.

However, the myeloma cells eventually acquire resistance to thosetherapeutic agents in most cases. Accordingly, the reality of thecurrent treatment means is that a myeloma patient has an unpromisingprognosis with a mean survival period after onset of from about 3 yearsto about 5 years. In addition, those therapeutic agents do notspecifically act on only tumor cells serving as targets, and hence havea problem of showing toxicity also to normal cells, consequently causingserious side effects.

There have been attempts to develop a treatment method for multiplemyeloma utilizing a monoclonal antibody. For example, an anti-CS1antibody, and an anti-CD38 antibody, and the like are consideredpromising (Non Patent Literatures 1 and 2). In addition, in PatentLiterature 1, there is disclosed a therapeutic agent for multiplemyeloma or the like, which uses an anti-human CD48 monoclonal antibodyas an active ingredient.

Integrins mainly form a heterodimer of an α-chain and a β-chain to servea function as a receptor on a cell surface in a living body. There aremany combinations of α-chains and β-chains of such integrins.

In addition, in Non Patent Literatures 4 to 6, there are disclosedchimeric antigen receptor T-cells (CAR-T cells) including an antigenrecognition site having an affinity for a certain antigen.

CITATION LIST Patent Literature

-   PTL 1: WO 2010/117059 A1

Non-patent Literature

-   NPL 1: Journal of Clinical Oncology, 2012 Jun. 1; 30(16): 1953-9.-   NPL 2: Journal of immunology, 2011 Feb. 1; 186(3): 1840-8.-   NPL 3: J Biol Chem. 2012 May 4; 287(19): 15749-59.-   NPL 4: J Immunol. 2009 Nov. 1; 183(9): 5563-74.-   NPL 5: N Engl J Med. 2014 Oct. 16; 371(16): 1507-17.-   NPL 6: Nat Biotechnol. 2002 Jan.; 20(1): 70-5.

SUMMARY OF INVENTION Technical Problem

The anti-CS1 antibody has relatively high specificity to myeloma cells.However, the antibody alone cannot be said to have a high anti-myelomaeffect, and its effectiveness as a single agent has not beendemonstrated in a clinical test. It has been found that the anti-tumoreffect of the anti-CS1 antibody is increased through combined use withlenalidomide, and it is considered that an approval is being sought forthe combined use. Meanwhile, CD38 is also expressed in many normal bloodcells including CD34-positive hematopoietic progenitor cells, and henceis an antigen having low specificity as a therapeutic target of multiplemyeloma. Under such circumstances, an object of the present invention isto provide means that is more effective for the treatment of, forexample, a disease involving neoplastic growth of plasma cells, such asmultiple myeloma.

Solution to Problem

The inventors of the present invention have made extensiveinvestigations in order to achieve such object, and as a result, haveobtained an MMG49 antibody by performing screening through use ofspecific binding to myeloma cells and progenitors thereof as anindicator. In addition, the inventors have confirmed that such antibodybinds to a certain region of human integrin β₇, and have found thatCAR-T cells generated using an antigen recognition site of such antibodyare extremely useful for the treatment of myeloma. In addition, theinventors have also elucidated that an epitope of the MMG49 antibody ispresent in the region of the amino acid residue positions 20 to 109 ofthe human integrin β₇.

The present invention has been completed on the basis of such findings,and encompasses inventions of a wide range of aspects described below.

(I) Antibody

An antibody (I) encompasses antibodies described in the following items(I-1) to (1-25).

(I-1)

An anti-human integrin β₇ antibody, whose epitope is present in a regionof the amino acid residue positions 20 to 109 of human integrin β₇.

(I-1A)

An antibody according to the item (I-1), whose epitope is present in aregion of the amino acid residue positions 33 to 109 of the humanintegrin β₇.

(I-1B)

An antibody according to the item (I-1), whose epitope is present in aregion of the amino acid residue positions 20 to 90 of the humanintegrin β₇.

(I-1C)

An antibody according to the item (I-1), whose epitope is present in aregion of the amino acid residue positions 33 to 90 of the humanintegrin β₇.

(I-2)

An antibody according to the item (I-1), whose affinity for the epitopeis increased in the presence of at least part of a region of the aminoacid residue positions 379 to 721 of the human integrin β₇.

(I-3)

An antibody according to the item (I-2), whose affinity for the epitopeis increased in the presence of at least part of a region of the aminoacid residue positions 417 to 721 of the human integrin β₇.

(I-4)

An antibody according to the item (I-2), whose affinity for the epitopeis increased in the presence of at least part of a region of the aminoacid residue positions 564 to 721 of the human integrin β₇.

(I-5)

An antibody according to the item (I-2), whose affinity for the epitopeis increased in the presence of at least part of a region of the aminoacid residue positions 379 to 563 of the human integrin β₇.

(I-6)

An antibody according to the item (I-2), whose affinity for the epitopeis increased in the presence of at least part of a region of the aminoacid residue positions 417 to 563 of the human integrin β₇.

(I-7)

An antibody according to the item (I-2), whose affinity for the epitopeis increased in the presence of at least part of a region of the aminoacid residue positions 379 to 416 of the human integrin β₇.

(I-8)

An antibody according to any one of the items (I-1) to (I-7), whoseaffinity for the epitope is increased through activation of the humanintegrin β₇.

(I-9)

An anti-human integrin β₇ antibody, whose affinity for human integrin β₇expressed on myeloma cells is higher than for human integrin β₇expressed on normal cells.

(I-10)

An antibody according to any one of the items (I-1) to (I-9), whoseepitope is identical to that of an MMG49 antibody.

(I-11)

An antibody according to any one of the items (I-1) to (I-10), theantibody including:

a heavy chain variable region including:

-   -   heavy-chain CDR1 having the amino acid sequence set forth in SEQ        ID NO: 1;    -   heavy-chain CDR2 having the amino acid sequence set forth in SEQ        ID NO: 2; and/or    -   heavy-chain CDR3 having the amino acid sequence set forth in SEQ        ID NO: 3; and/or a light chain variable region including:

light-chain CDR1 having the amino acid sequence set forth in SEQ ID NO:6;

-   -   light-chain CDR2 having the amino acid sequence set forth in SEQ        ID NO: 7; and/or    -   light-chain CDR3 having the amino acid sequence set forth in SEQ        ID NO: 8.

(I-12)

An antibody according to any one of the items (I-1) to (I-10), theantibody including:

a heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 4; and/or

a light chain variable region having the amino acid sequence set forthin SEQ ID NO: 9.

(I-13)

An antibody according to any one of the items (I-1) to (I-12), which isFv, scFv, a diabody, a triabody, a tetrabody, or a combination thereof.

(I-14)

An antibody according to any one of the items (I-1) to (I-11), theantibody including a constant region.

(I-15)

An antibody according to any one of the items (I-1) to (I-12) and(I-14), which is a chimeric antibody.

(I-16)

An antibody according to any one of the items (I-1) to (I-12) and(I-14), which is a humanized antibody.

(I-17)

An antibody according to any one of the items (I-1) to (I-12) and(I-14), which is a human antibody.

(I-18)

An antibody according to any one of the items (I-1) to (I-12) and (I-14)to (I-17), which is an immunoglobulin, Fab, F(ab′)2, a minibody,scFv-Fc, or a combination thereof.

(I-19)

An antibody according to any one of the items (I-1) to (I-12) and (I-14)to (I-18), which is IgA, IgD, IgE, IgG, or IgM.

(I-20) An antibody according to any one of the items (I-1) to (I-12) and(I-14) to (I-19), the antibody including a heavy chain having the aminoacid sequence set forth in SEQ ID NO: 5 and/or a light chain having theamino acid sequence set forth in SEQ ID NO: 10.

(I-21)

An antibody according to any one of the items (I-1) to (I-20), which hascytotoxic activity.

(I-22)

An antibody according to the item (I-21), in which the cytotoxicactivity is ADCC activity and/or CDC activity.

(1-23)

An antibody according to any one of the items (I-1) to (I-22), which isa multispecific antibody.

(I-24)

An antibody according to any one of the items (I-1) to (I-23), which hasa cytotoxin bound thereto.

(I-25)

An antibody according to any one of the items (I-1) to (I-24), which isa monoclonal antibody.

(II) Polynucleotide

A polynucleotide (II) encompasses a polynucleotide described in thefollowing item (II-1).

(II-1)

A polynucleotide, which has a base sequence encoding the amino acidsequence of the antibody (I).

(III) Host Cell

A host cell (III) encompasses a host cell described in the followingitem (III-1) or (III-2).

(III-1)

A host cell, which harbors the polynucleotide (II).

(III-2)

A host cell according to the item (III-1), which is a eukaryotic cell.

(IV) Chimeric Antigen Receptor

A chimeric antigen receptor (IV) encompasses chimeric antigen receptorsdescribed in the following items (IV-1) to (IV-5).

(IV-1)

A chimeric antigen receptor, whose epitope is identical to that of theantibody (I).

(IV-2)

A chimeric antigen receptor according to the item (IV-1), the chimericantigen receptor including an antigen recognition site of the antibody(I).

(IV-3)

A chimeric antigen receptor according to the item (IV-1) or (IV-2), theantigen recognition site including:

a heavy chain variable region including:

-   -   heavy-chain CDR1 having the amino acid sequence set forth in SEQ        ID NO: 1;    -   heavy-chain CDR2 having the amino acid sequence set forth in SEQ        ID NO: 2; and/or    -   heavy-chain CDR3 having the amino acid sequence set forth in SEQ        ID NO: 3; and/or

a light chain variable region including:

-   -   light-chain CDR1 having the amino acid sequence set forth in SEQ        ID NO: 6;    -   light-chain CDR2 having the amino acid sequence set forth in SEQ        ID NO: 7; and/or    -   light-chain CDR3 having the amino acid sequence set forth in SEQ        ID NO: 8.

(IV-4)

A chimeric antigen receptor according to any one of the items (IV-1) to(IV-3), in which the antigen recognition site includes:

a heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 4; and/or

a light chain variable region having the amino acid sequence set forthin SEQ ID NO: 9.

(IV-5)

A chimeric antigen receptor according to any one of the items (IV-1) to(IV-4), the chimeric antigen receptor having the amino acid sequence setforth in SEQ ID NO: 21.

(V) Polynucleotide

A polynucleotide (V) encompasses a polynucleotide described in thefollowing item (V-1) or (V-2) unlike the polynucleotide (II).

(V-1)

A polynucleotide, which encodes the amino acid sequence of the chimericantigen receptor (IV).

(V-2)

A polynucleotide according to the item (V-1), which has the basesequence set forth in SEQ ID NO: 22.

(VI) Cell

A cell (VI) encompasses a cell described in any one of the followingitems (VI-1) to (VI-4) unlike the host cell (III).

(VI-1)

A cell, which harbors the polynucleotide (V).

(VI-2)

A cell according to the item (VI-1), which is a eukaryotic cell.

(VI-3)

A cell according to the item (VI-1) or (VI-2), which is a T-cell or anNK cell.

(VI-4)

A cell according to any one of the items (VI-1) to (VI-3), which is achimeric antigen receptor T-cell or a chimeric antigen receptor NK cell.

(VII) Pharmaceutical Composition

A pharmaceutical composition (VII) encompasses pharmaceuticalcompositions described in the following items (VII-1) to (VII-5).

(VII-1)

A pharmaceutical composition, including the antibody (I) or the cell(VI).

(VII-2)

A pharmaceutical composition according to the item (VII-1), in which thecell is the chimeric antigen receptor T-cell (VI-4).

(VII-3) A pharmaceutical composition according to the item (VII-1) or(VII-2), which is for use in treatment of cancer.

(VII-4)

A pharmaceutical composition according to the item (VII-3), in which thecancer is blood cancer.

(VII-5)

A pharmaceutical composition according to the item (VII-4), in which theblood cancer is a disease causing neoplastic growth of plasma cells.

(VIII) Treatment or Prevention Method for Disease

A treatment or prevention method (VIII) for a disease encompassestreatment or prevention methods for a disease described in the followingitems (VIII-1) to (VIII-6).

(VIII-1)

A treatment or prevention method for a disease, including administeringa therapeutically effective amount of the antibody (I) or the cell (VI)to a subject.

(VIII-2)

A treatment or prevention method according to the item (VIII-1), inwhich the cell is the chimeric antigen receptor T-cell (VI-4).

(VIII-3)

A treatment or prevention method according to the item (VIII-1) or(VIII-2), in which the disease is cancer, and in which the subject is apatient who has developed cancer or an animal having a risk ofdeveloping cancer.

(VIII-4) A treatment or prevention method according to the item(VIII-3), in which the cancer is blood cancer.

(VIII-5)

A treatment or prevention method according to the item (VIII-4), inwhich the blood cancer is a disease causing neoplastic growth of plasmacells.

(VIII-6)

A treatment or prevention method for multiple myeloma, targetingactive-form human integrin β₇.

(IX) Use

A use (IX) encompasses uses described in the following items (IX-1) to(IX-5).

(IX-1)

A use of the antibody (I) or the cell (VI), for producing apharmaceutical composition.

(IX-2)

A treatment or prevention method according to the item (IX-1), in whichthe cell is the chimeric antigen receptor T-cell (VI-4).

(IX-3)

A use according to the item (IX-1) or (IX-2), which is for treatment ofcancer.

(IX-4)

A use according to the item (IX-3), in which the cancer is blood cancer.

(IX-5)

A use according to the item (IX-4), in which the blood cancer is adisease causing neoplastic growth of plasma cells.

(X) Screening Method

A screening method (X) encompasses screening methods described in thefollowing (X-1) to (X-5).

(X-1)

A screening method for an active ingredient of a pharmaceuticalcomposition for treating or preventing cancer, the method includingselecting, from a compound library, a candidate substance thatspecifically binds to human integrin β₇ and binds to a region of theamino acid residue positions 20 to 109 of the human integrin β₇.

(X-2)

A screening method according to the item (X-1), further includingselecting a substance having cytotoxic activity.

(X-3)

A screening method according to the item (X-1) or (X-2), in which thesubstance to be selected is a monoclonal antibody.

(X-4)

A screening method according to any one of the items (X-1) to (X-3), inwhich the cancer is blood cancer.

(X-5)

A screening method according to the item (X-4), in which the bloodcancer is a disease causing neoplastic growth of plasma cells.

(XI) Diagnosis Method

A diagnosis method (XI) encompasses diagnosis methods described in thefollowing items (XI-1) to (XI-5).

(XI-1)

A diagnosis method for cancer, including bringing a sample collectedfrom a subject into contact with the antibody (I).

(XI-2)

A diagnosis method according to the item (XI-1), in which the samplecollected from a subject is blood or bone marrow fluid.

(XI-3)

A diagnosis method according to the item (XI-1) or (XI-2), furtherincluding judging that the subject has developed, or has a risk ofdeveloping, cancer when cells that bind to the antibody (I) aredetected.

(XI-4)

A diagnosis method according to the item (XI-3), in which the cancer isblood cancer.

(XI-5)

A diagnosis method according to the item (XI-4), in which the cells areplasma cells, and in which the cancer is a disease causing neoplasticgrowth of plasma cells.

(XII) Kit

A kit (XII) encompasses kits described in the following items (XII-1) to(XII-3).

(XII-1)

A kit for diagnosis of cancer, including the antibody (I).

(XII-2)

A diagnosis method according to the item (XII-1), in which the cancer isblood cancer.

(XII-3)

A kit according to the item (XII-2), in which the cancer is a diseasecausing neoplastic growth of plasma cells.

Advantageous Effects of Invention

The antibody of the present invention does not recognize normal cells,and hence is useful as an active ingredient of a pharmaceuticalcomposition. In particular, the antibody of the present invention isuseful as an active ingredient of a therapeutic agent for cancer (e.g.,blood cancer).

The antibody of the present invention is useful because chimeric antigenreceptor T-cells produced by applying its antigen recognition site to achimeric antigen receptor can be used as an active ingredient of suchpharmaceutical composition as described above.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results obtained in Example 2 by analyzing the binding ofan MMG49 antibody to myeloma patient-derived bone marrow cells throughuse of FACS. (Left) A diagram for illustrating a method of identifying amyeloma progenitor cell fraction (Myeloma progenitor cells), a myelomaplasma cell fraction (Myeloma plasma cells), and CD45⁺ leukocytes (CD45⁺leukocytes). (Right) Graphs for showing the binding of the MMG49antibody to each of the fractions.

FIG. 2 shows graphs for results obtained in Example 2 by analyzing, byFACS, the binding of the MMG49 antibody to the myeloma progenitor cellfraction, myeloma plasma cell fraction, and CD45⁺ leukocytes of aplurality of myeloma patient-derived bone marrow cells (UPN1 to UPN5).

FIG. 3 is an illustration of a process of identifying an antigen proteinrecognized by the MMG49 antibody by an expression cloning method inExample 3. There is illustrated a process of concentrating BaF3 cellsthat bind to the MMG49 antibody, from an initial concentration of 0.1%or less, by FACS sorting.

FIG. 4 shows graphs for results obtained in Example 4 by stainingITGB7-deficient U266 cells generated using a Crisp-cas9 system with theMMG49 antibody or an FIB27 antibody (commercially availableanti-integrin β₇ antibody), followed by FACS analysis.

FIG. 5 is an image for showing results obtained in Example 4 bysubjecting a product immunoprecipitated from a cell lysate derived fromMM1s myeloma cells with the MMG49 antibody or an isotype controlantibody, to SDS-PAGE, and then performing western blot with acommercially available anti-integrin β₇ antibody (Abcam plc).

FIGS. 6A and 6B are graphs showing results obtained in Example 5 byanalyzing the binding of each of the MMG49 antibody, the FIB27 antibody,and an FIB504 antibody to each cell fraction of healthy personperipheral blood cells (in the figures, B-cells, T-cells, monocytes,neutrophils, red blood cells, and platelets are shown in the statedorder from the left-hand side) through use of FACS.

FIGS. 7A and 7B are graphs showing results obtained in Example 5 byanalyzing, by FACS, the binding of the MMG49 antibody to each of cellfractions of myeloma patient-derived bone marrow cells. On the left-handside, a method of identifying each cell fraction is illustrated, and onthe right-hand side, graphs for showing the binding of MMG49 to eachfraction are shown. In FIG. 7A, a comparison between hematopoietic stemcell and progenitor cell fractions, and myeloma cells is shown, and inFIGS. 7B, a comparison between B/T lymphocyte fractions, and myelomaprogenitor cell and myeloma plasma cell fractions is shown.

FIG. 8 is graphs showing results obtained in Example 6 by analyzing thebinding of each of the MMG49 antibody and the FIB27 antibody to each ofvarious myeloma cell lines, and T-cells and B-cells derived fromperipheral blood through use of FACS. There are also shown results ofconfirming the expression of ITGA4 (binding of an anti-integrin α4antibody) and the expression of ITGAE (binding of an anti-integrin α_(E)antibody) in the above-mentioned cells by FACS analysis.

FIG. 9 is graphs showing results obtained in Example 6 by analyzing, byFACS, the binding of the MMG49 antibody and the FIB27 antibody to U266cells and ITGA4 (integrin α4)-deficient U266 cells. There are also shownresults obtained by analyzing, by FACS, the expression of ITGA4 (bindingof the anti-integrin α4 antibody) in the above-mentioned cells.

FIGS. 10A and 10B are graphs showing results obtained in Example 7 byallowing integrin α₄β₇-forcibly expressing K562 cells and human normalperipheral blood-derived T-cells treated in the presence of Ca²⁺/Mg²⁺ orMn²⁺ at 37° C. for 20 minutes to react with the MMG49 antibody or anisotype antibody, then staining the cells using an anti-mouse IgGantibody as a secondary antibody, and subjecting the stained cells toFACS analysis.

FIG. 11 is a diagram illustrating the construction of human/mousechimeric integrin β₇ proteins and the presence or absence of the bindingof the MMG49 antibody to 293T cells caused to transiently express theproteins in Example 8.

FIGS. 12A and 12B are graphs showing results obtained in Example 8 byanalyzing, by FACS, the binding of the MMG49 antibody to 293T cellscaused to transiently express the human/mouse chimeric integrin β₇proteins.

FIG. 13 is a graph summarizing the results shown in FIG. 12. In thegraph in FIG. 13, the axis of ordinate represents the percentage ofcells bound to the antibody, and the axis of abscissa represents varioushuman/mouse chimeric integrin β₇ proteins.

FIG. 14 is graphs showing results obtained by staining MM1s cells andKMS12BM cells with a chimerized MMG49 antibody generated by linkingvariable regions of the MMG49 antibody to a human IgG4 antibody constantdomain.

FIG. 15 is a scheme illustrating a method of generating a CAR constructusing variable regions of the MMG49 antibody.

FIG. 16 is graphs showing results obtained by staining T-cells caused toexpress the CAR construct using variable regions of the MMG49 antibodywith a PE-anti-human F(ab′)2 antibody.

FIG. 17 is graphs showing results obtained in Example 11 byquantitatively determining, by ELISA, the amounts of IFN-γ and IL2produced through coculture of MMG49 antibody-derived CAR-T cells orT-cells having introduced therein GFP (control) with K562 cellsexpressing no integrin β₇ or K562 cells caused to forcibly expressintegrin α4137. *: p<0.05.

FIG. 18 is graphs showing results obtained in Example 11 byquantitatively determining, by ELISA, the amount of IFN-γ producedthrough coculture of MMG49 antibody-derived CAR-T cells or T-cellshaving introduced therein GFP (control) with MMG49 antigen-expressingcells or non-expressing cells.

FIG. 19 is graphs showing results obtained in Example 11 byquantitatively determining, by ELISA, the amount of IL2 produced throughcoculture of MMG49 antibody-derived CAR-T cells or T-cells havingintroduced therein GFP (control) with MMG49 antigen-expressing cells ornon-expressing cells.

FIG. 20 is graphs showing results obtained in Example 11 by measuring,by ⁵¹Cr killing assay, the degree of cell damage caused by MMG49antibody-derived CAR-T cells or T-cells having introduced therein GFP(control) with respect to K562 cells expressing no integrin β₇ or K562cells caused to forcibly express integrin α4137. The y-axis of each ofthe graphs in FIG. 20 represents a cell damage percentage (%).

FIG. 21 is graphs showing results obtained in Example 11 by measuring,by ⁵¹Cr killing assay, the degree of cell damage caused by MMG49antibody-derived CAR-T cells or T-cells having introduced therein GFP(control) with respect to MMG49 antigen-expressing cells ornon-expressing cells.

FIG. 22 is a diagram and graphs for illustrating and showing the designof a therapeutic experiment for a myeloma cell line MM1s engrafted inthe bone marrow of an NOG mouse and results thereof in Example 12. Bonemarrow cells after 1 week from the transfer of MMG49 antibody-derivedCAR-T cells or T-cells having introduced therein GFP (control) werecollected and analyzed by FACS. MM1s cells can be identified as humanCD138⁺ cells. In an MMG49 antibody-derived CAR-T cell-administeredgroup, MM1s cells in the bone marrow have almost completely disappeared.

FIGS. 23A and 23B are a diagram, images, and a graph for illustratingand showing the design of a therapeutic experiment for the myeloma cellline MM1s systemically engrafted to an NOG mouse and results thereof inExample 12. The amounts of myeloma cells before and after the transferof MMG49 antibody-derived CAR-T cells or T-cells having introducedtherein GFP (control) were evaluated by fluorescence intensitymeasurement based on IVIS imaging. In an MMG49 antibody-derived CAR-Tcell-administered group, MM1s cells in the bone marrow have almostcompletely disappeared.

FIGS. 24A and 24B are views for illustrating a comparison between theamino acid sequence of integrin β₇ of human origin and the amino acidsequence of integrin β₇ of mouse origin.

FIG. 25 is a diagram and graphs for illustrating and showing theconstruction of human/mouse chimeric integrin β₇ proteins and thepresence or absence of the binding of the MMG49 antibody to 293T cellscaused to transiently express the proteins in Example 13.

FIG. 26 is a graph for showing results of an experiment forinvestigating an epitope of the MMG49 antibody in Example 14. MFI on theaxis of abscissa represents binding strength to the MMG49 antibody, anda higher numerical value indicates a higher avidity.

DESCRIPTION OF EMBODIMENTS

Herein, “include” and “have” are so-called open language, but are each aconcept including the closed language “consisting of”, and in oneembodiment, may be replaced by “consisting of”.

A “myeloma progenitor cell” is a progenitor cell in a stage beforedifferentiating into a myeloma plasma cell, and is characterized byhighly expressing CD38, but not expressing CD138, which serves as amarker specific to a mature plasma cell.

Therefore, the myeloma progenitor cell is sometimes referred to as“CD38⁺+CD138⁻ cell” or “CD19⁻CD38⁺+CD138⁻ cell”.

A “myeloma plasma cell” is generally also called a myeloma cell, and isa cell that produces M protein, which is an abnormal immunoglobulin. Themyeloma plasma cell expresses CD138 in addition to highly expressingCD38. Therefore, the myeloma plasma cell is sometimes referred to as“CD38⁺⁺CD138⁺ cell” or “CD19⁻CD38⁺⁺CD138⁺ cell”.

The myeloma progenitor cell and the myeloma plasma cell also mean atumor progenitor cell and a neoplastic plasma cell, respectively, in adisease causing neoplastic growth of plasma cells other than multiplemyeloma.

A “hematopoietic progenitor cell” is a cell capable of differentiatinginto various hematopoietic cells. The hematopoietic progenitor cell ischaracterized by expressing CD34. Therefore, herein, the hematopoieticprogenitor cell is sometimes referred to as “CD34⁺ cell”.

(I) Antibody

An antibody (I) is preferably an anti-human integrin β₇ antibody whoseepitope is present in the region of the amino acid residue positions 20to 109 of human integrin β₇.

More preferred examples of the antibody (I) may include: an antibodywhose epitope is present in the region of the amino acid residuepositions 33 to 109 of human integrin β₇; and an antibody whose epitopeis present in the region of the amino acid residue positions 20 to 90 ofhuman integrin β₇. The most preferred example thereof may be an antibodywhose epitope is present in the region of the amino acid residuepositions 33 to 90 of human integrin β₇.

The human integrin β₇ is not particularly limited, and may be atransmembrane protein having the amino acid sequence set forth in SEQ IDNO: 31, the protein being capable of forming a heterodimer with integrinα. Specific examples of the integrin α may include integrin α4 andintegrin α_(E).

Specific examples of the amino acid sequence of the human integrin β₇may include, in addition to the amino acid sequence set forth in SEQ IDNO: 31, amino acid sequences described in, for example: ACCESSION:EAW96675, VERSION: EAW96675.1, GI: 119617081; ACCESSION: NM000889,VERSION: NM000889.2, GI: 540344585; ACCESSION: XM005268851, VERSION:XM005268851.2, GI: 767974096; ACCESSION: XM006719376, VERSION:XM006719376.2, GI: 767974098; and ACCESSION: XM005268852, VERSION:XM005268852.3, GI: 767974097, listed in the NCBI database.

The following description regarding the human integrin β₇ is made on thebasis of the amino acid sequence set forth in SEQ ID NO: 31. However,for any other amino acid sequence of the human integrin β₇, a personskilled in the art can easily judge which region or site of the otheramino acid sequence of the human integrin β₇ corresponds to a regionand/or site of the human integrin β₇ to be described below bydetermining the homology of the other amino acid sequence to the aminoacid sequence set forth in SEQ ID NO: 31 in silico.

The region of the amino acid residue positions 1 to 19 of the humanintegrin β₇ is a peptide fragment serving as a signal peptide and beingabsent when the human integrin β₇ functions as a membrane protein in aliving body. Accordingly, when the human integrin β₇ exhibits a functionas a membrane protein, its N-terminus is the amino acid residue atposition 20 of the above-mentioned amino acid sequence.

The region of the amino acid residue positions 20 to 109 of the humanintegrin β₇ includes a PSI domain. The PSI domain of the human integrinβ₇ and the PSI domain of mouse integrin β₇ are known to have a highhomology of about 80% or more. However, as illustrated in FIG. 24, whencompared to each other, the amino acid residues of the regions of theamino acid residue positions 20 to 109 including the PSI domains of thehuman integrin β₇ and the mouse integrin β₇ differ from each other at atotal of 15 amino acid residues, specifically amino acid residues atposition 23, position 26, position 28, position 30, position 32,position 35, position 36, position 38, position 41, position 42,position 48, position 93, position 94, position 102, and position 109 ofthe human integrin β₇.

Therefore, it is preferred that the epitope of the antibody (I) beassociated with any one or more, preferably two or more, more preferablythree or more of those 15 amino acid residues.

Specifically, the epitope of the antibody (I) is preferably present inthe region of the amino acid residue positions 23 to 109 of the humanintegrin β₇, more preferably present in the region of the amino acidresidue positions 23 to 48 or the region of the amino acid residuepositions 93 to 109.

The epitope of the antibody (I) in another more preferred embodiment maybe: the region of the amino acid residue positions 23 to 48; the regionof the amino acid residue positions 93 to 109; or a three-dimensionalregion that is a combination of the region of the amino acid residuepositions 23 to 48 and the region of the amino acid residue positions 93to position 109.

The epitope of the antibody (I) may be a linear epitope, or may be aconformational epitope (also called a non-linear epitope). It is knownto a person skilled in the art that the linear epitope is a case inwhich consecutive amino acid residues serve as an epitope and theconformational epitope is an epitope formed of non-consecutive aminoacid residues.

For example, the case in which the above-mentioned three-dimensionalregion that is a combination of the region of the amino acid residuepositions 23 to 48 and the region of the amino acid residue positions 93to 109 serves as the epitope may be given as an example corresponding tothe conformational epitope, and a case in which a region ofnon-consecutive amino acid residues included in the region of the aminoacid residue positions 20 to 109 serves as the epitope is alsoencompassed in the conformational epitope.

Of the above-mentioned epitopes, it is preferred that the amino acidresidue at position 48 be strongly related to the epitope of theantibody (I) or be included in the epitope of the antibody (I).

A person skilled in the art can understand about specific linearepitopes and conformational epitopes with reference to, for example, JP2011-527572 A, JP 2009-534401 A, or “Dissecting antibodies with regardsto linear and conformational epitopes.” Forsstrom B, Axnas BB, RockbergJ, Danielsson H, Bohlin A, Uhlen M. PLoS One. 2015 Mar. 27; 10(3):e0121673. doi: 10.1371/journal.pone.0121673. eCollection 2015.

In other words, the foregoing means that the antibody (I) is an antibodythat specifically binds to the region of the amino acid residuepositions 20 to 109 of the human integrin β₇, and in particular,preferably specifically binds to the region at positions from 23 to 109,more preferably specifically binds to the region at positions from 23 to48 and/or positions from 93 to 109.

In addition, the property of the antibody (I) of binding to the regionof the amino acid residue positions 20 to 109 of the integrin β₇, whichserves as the epitope, is sometimes referred to as affinity for theepitope. Accordingly, the term “affinity for the epitope is increased”has the same meaning as “specific binding capacity for the epitope isincreased.”

The term “specific” may be distinguished from the term “selective”.

As another embodiment of the antibody (I), it is preferred that theaffinity of the antibody (I) for the epitope be increased in thepresence of at least part of the region of the amino acid residuepositions 379 to 721 of the human integrin β₇.

The “at least part of the region of the amino acid residue positions 379to 721” means that any one of the region of the amino acid residuepositions 379 to 721 and a partial region thereof may be adopted.Specific examples of the “partial region thereof” include: at least partof the region of the amino acid residue positions 417 to 721 of thehuman integrin β₇; at least part of the region of the amino acid residuepositions 564 to 721 of the human integrin β₇; at least part of theregion of the amino acid residue positions 379 to 563 of the humanintegrin β₇; at least part of the region of the amino acid residuepositions 417 to 563 of the human integrin β₇; and at least part of theregion of the amino acid residue positions 379 to 416 of the humanintegrin β₇. That is, the affinity of the antibody (I) for the epitopecan be increased in the presence of any of those regions.

The term “in the presence of” means that the region of the amino acidresidue positions 20 to 109 of the human integrin β₇ and at least partof the region of the amino acid residue positions 379 to 721 of thehuman integrin β₇ may be present in the same molecule, or the tworegions may be present as separate molecules. It is preferred that thetwo regions be present in the same molecule. The term “in the presenceof” may be read as “by”.

A person skilled in the art can easily confirm that the affinity of theabove-mentioned antibody (I) for the epitope is increased, by a commonlyused immunoassay method described in, for example, Examples to bedescribed below.

For example, cells caused to express a human/mouse chimeric integrin β₇protein (#4960), which is various human/mouse chimeric integrin β₇proteins described in Example 8, and which includes the region of theamino acid residue positions 1 to 109 of integrin β₇ of human origin andincludes the region of the amino acid residue positions 722 to 798 ofthe integrin β₇ of human origin, are prepared, and a human/mousechimeric integrin β₇ protein (#4961) in which the region of #4960 of theamino acid residue positions 379 to 721 of the integrin β₇ of humanorigin is replaced with the region of the amino acid residue positions379 to 721 of integrin β₇ of mouse origin is prepared. In this case, theincrease in the affinity of the antibody (I) for the epitope may beconfirmed by comparing the degrees of binding of the antibody (I)between cells expressing the latter (#4961) and cells expressing theformer (#4960).

As another embodiment of the antibody (I), it is preferred that theaffinity of the antibody (I) for the epitope be increased by activatingthe human integrin β₇. Probably because activated human integrin β₇ hasa structural feature in a region including the epitope, the affinity ofthe antibody (I) for the epitope is increased.

A method of activating the human integrin β₇ is known. For example, byallowing a phorbol ester, such as PMA, a manganese salt, or the like toact on cells expressing the human integrin β₇, e.g., cells selected fromblood cells and immune cells, such as plasma cells, NK cells, T-cells,B-cells, lymphoblasts, Burkitt lymphoma-derived cells, and dendriticcells, the human integrin β₇ expressed in the cells may be activated. Inaddition, without being limited to the above-mentioned specific cells,cells caused to express the human integrin β₇ may be used and treatedwith a phorbol ester, a manganese salt, or the like to activate thehuman integrin β₇.

A person skilled in the art can easily confirm that the affinity of theantibody (I) for the epitope is increased through activation of thehuman integrin β₇, by a commonly used immunoassay method described in,for example, Examples to be described below.

For example, cells caused to express #4960 or #4961, which is thevarious human/mouse chimeric integrin β₇ proteins described in Example 8and includes the region of the amino acid residue positions 1 to 109,are prepared, and the cells are subjected to integrin β₇-activatingmeans as described in Example 7. After that, affinities before and afterthe activation treatment are compared to each other through measurementusing immunoassay means. Thus, the increase in the affinity of theantibody (I) for the epitope in the cells after the activation may beconfirmed.

As another embodiment of the antibody (I), the antibody (I) may be ananti-human integrin β₇ antibody having a feature of having a higheraffinity for human integrin β₇ expressed on myeloma-derived cells thanfor human integrin β₇ expressed on normal cells.

The normal cells are not particularly limited as long as the cells arederived from a healthy person, and may be, for example, blood-derivednormal cells. Of such normal cells, normal plasma cells are preferred.

A method of confirming that the antibody has a higher affinity for humanintegrin β₇ expressed on myeloma cells than for human integrin β₇expressed on such normal cells can easily be performed by a personskilled in the art by a commonly used immunoassay method described in,for example, Examples to be described below.

The “commonly used immunoassay method” is not particularly limited aslong as the method involves measurement using various antibodiesirrespective of the antigen. Examples thereof may include a flowcytometry method (FACS), cell sorting involved therein, westernblotting, ELISA, an immunoprecipitation method, a SPR method, and a QCMmethod.

As another embodiment of the antibody (I), an epitope of the antibody(I) is preferably identical to that of an MMG49 antibody disclosed inExamples to be described later. An antibody identical to the MMG49antibody is most preferred. For a method of producing the MMG49antibody, reference may be made to Examples to be described below.

As another embodiment of the antibody (I), the antibody (I) ispreferably an antibody of an embodiment including a heavy chain variableregion and/or a light chain variable region. That is, the antibody (I)may be the heavy chain variable region alone, or may be the light chainvariable region alone. The antibody (I) is preferably an antibodyincluding the heavy chain variable region and the light chain variableregion.

A variable region is also called an antigen recognition site, and isunderstood by a person skilled in the art to be a site important for anantibody to recognize an antigen. Such variable region has three regionscalled hypervariable regions (also referred to as complementaritydetermining regions [CDRs]), and it is also known to a person skilled inthe art that the CDRs are extremely important regions most involved inthe antigen recognition function of an antibody.

The heavy chain variable region included in the other embodiment of theantibody (I) includes any one or more of heavy-chain CDR1, heavy-chainCDR2, and heavy-chain CDR3. That is, the heavy chain variable region maycontain heavy-chain CDR1, heavy-chain CDR2, or heavy-chain CDR3 alone,and preferably includes at least heavy-chain CDR3. A more preferredembodiment includes heavy-chain CDR1, heavy-chain CDR2, and heavy-chainCDR3 in the stated order from the amino-terminus (N-terminus).

The light chain variable region may be similar to the heavy chainvariable region, i.e., includes, for example, any one of light-chainCDR1, light-chain CDR2, and light-chain CDR3, preferably includes atleast light-chain CDR3, and preferably includes light-chain CDR1,light-chain CDR2, and light-chain CDR3 in the stated order from theN-terminus of the light chain variable region.

Regions other than CDR1 to CDR3 in each of the heavy chain variableregion and the light chain variable region are sometimes referred to asFRs. More specifically, a region between the N-terminus and the CDR1 iscalled FR1, a region between the CDR1 and the CDR2 is called FR2, aregion between the CDR2 and the CDR3 is called FR3, and a region betweenthe CDR3 and the carboxy-terminus (C-terminus) is called FR4, and thenames are designated for each of the heavy chain variable region and thelight chain variable region.

The amino acid sequences of the heavy-chain CDR1 to CDR3 and thelight-chain CDR1 to CDR3 are not particularly limited. Examples thereofinclude heavy-chain CDR1 having the amino acid sequence set forth in SEQID NO: 1, heavy-chain CDR2 having the amino acid sequence set forth inSEQ ID NO: 2, heavy-chain CDR3 having the amino acid sequence set forthin SEQ ID NO: 3, light-chain CDR1 having the amino acid sequence setforth in SEQ ID NO: 6, light-chain CDR2 having the amino acid sequenceset forth in SEQ ID NO: 7, and light-chain CDR3 having the amino acidsequence set forth in SEQ ID NO: 8 serving as heavy-chain CDRs 1 to 3 orlight-chain CDRs 1 to 3 of the MMG49 antibody.

As a preferred embodiment of the heavy chain variable region includingthe heavy-chain CDR1 to CDR3, there may be given, for example, a heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 4, which is a heavy chain variable region of the MMG49 antibody. Inaddition, as a preferred embodiment of the light chain variable regionincluding the light-chain CDR1 to CDR3, there may be given, for example,a light chain variable region having the amino acid sequence set forthin SEQ ID NO: 9, which is a light chain variable region of the MMG49antibody.

The above-mentioned amino acid sequences of the MMG49 antibody set forthin SEQ ID NOS: 1 to 4 and 6 to 9 are as shown in Table 1 below.Underlined parts in each of the amino acid sequences of the heavy chainand variable regions set forth in SEQ ID NOS: 4 and 9 in Table 1indicate portions located at the CDR1, the CDR2, and the CDR3 in thestated order from the N-terminus.

TABLE 1 <Amino acid sequences of MMG49 antibody> HeavyCDR1 (SEQ ID NO: 1) GYTFSSYW chain CDR2 (SEQ ID NO: 2) MLPGSGSSCDR3 (SEQ ID NO: 3) ARGDGNYWYFDV Variable regionMEWTWVFLFLLSVTAGVESQVQLQQSGAELMKPGASVK (SEQ ID NO: 4)ISCKASGYTFSSYWIEWVKQRPGHGLEWIGEMLPGSGSSNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYY CARGDGNYWYFDVWGAG LightCDR1 (SEQ ID NO: 6) SSVGY chain CDR2 (SEQ ID NO: 7) ATSCDR3 (SEQ ID NO: 8) QQWSSDPPT Variable regionMDFQVQIFSFLLISASVIMSRGQIVISQSPAILSASPG (SEQ ID NO: 9)EKVTMTCRASSSVGYMHWFQQKPGSSPKPWIYATSNLASGVPARFSGSESGTSYSLTISRVEAEDAATYYCQQWSS DPPTFGGGTKLEIK

The structure of the antibody (I) is not limited. Specific examples ofthe structure include Fv, scFv, a diabody, a triabody, and a tetrabody,and the structure may also be a structure obtained by appropriatelycombining these structures. In addition, those structures including thecombined structures as well are each sometimes also called a fragmentantibody. Such fragment antibody may be an artificially designedrecombinant protein including Fv, or may be one fused with abiomolecule, such as a protein.

The Fv is also called the smallest structural unit of an antibody, andis a structure in which a heavy chain variable region and a light chainvariable region are associated with each other through a non-covalentintermolecular interaction. Further, the Fv may be a structure in whichthe thiol groups of cysteine residues present in the heavy chainvariable region and the light chain variable region form a disulfidebond with each other.

The scFv is a structure in which the C-terminus of a heavy chainvariable region and the N-terminus of a light chain variable region arelinked through a linker, and is also called a single-chain antibody. Inaddition, the C-terminus and N-terminus to be linked through a linkermay be the C-terminus of the light chain variable region and theN-terminus of the heavy chain variable region. The structure of the scFvmay be formed by association based on a non-covalent intermolecularinteraction or the like as in the Fv.

The diabody, the triabody, and the tetrabody are structures in which theabove-mentioned scFv forms a dimer, a trimer, and a tetramer,respectively, and is associated in the structurally most stable statethrough a non-covalent intermolecular interaction or the like betweenvariable regions as in the Fv or the like.

A person skilled in the art can easily produce the antibody (I) havingany of such various structures by: constructing an expression vectorthrough use of commonly used genetic engineering means; and using, withsuch expression vector, an expression system adopting host cells suitedfor antibody production, such as prokaryotic cells (such as Escherichiacoli or actinomycetes) or eukaryotic cells (such as yeast cells, insectcells, or mammalian cells), a commonly used cell-free expression system,or the like. The produced antibody may be appropriately subjected to acommonly used purification process so as to be obtained in a high-puritystate.

As another embodiment of the antibody (I), the antibody (I) may containa constant region. The constant region is understood by a person skilledin the art to be as follows: a heavy chain constant region includes CH1,CH2, and CH3, and a light chain constant region includes CL. Inaddition, a region including CH2 and CH3 is sometimes called an Fcdomain.

A specific origin of the constant region is not particularly limited.Examples thereof may include constant regions originating from animalspecies capable of mass production, animal species closely related tohumans, animal species that are less liable to cause immunogenicity inadministration to a human, and the like, e.g., constant regions of humanorigin, mouse origin, rat origin, rabbit origin, monkey origin, andchimpanzee origin.

In the antibody (I), when the heavy chain variable region and/or thelight chain variable region have an amino acid sequence of mouse origin,for example, a constant region of human origin may be combinedtherewith, to thereby provide the antibody (I) as a chimeric antibody.

In addition, heavy-chain FR1 to FR4 and/or light-chain FR1 to FR4 in theabove-mentioned chimeric antibody may be replaced with amino acidsequences of human origin, to thereby provide the antibody (I) as ahumanized antibody.

Further, heavy-chain CDR1 to CDR3 and/or light-chain CDR1 to CDR3 in theabove-mentioned humanized antibody may be replaced with amino acidsequences of human origin to the extent that the functions of the CDRsare not reduced, to thereby provide the antibody (I) as a humanantibody. The term “human antibody” is sometimes called a “completelyhumanized antibody”.

Examples of the structure of the antibody (I) of the embodimentincluding a constant region may include structures such as Fab, F(ab′)2,a minibody, and scFv-Fc, as well as an immunoglobulin having afour-chain structure including a pair of heavy chains each having aheavy chain variable region and a heavy chain constant region, and apair of light chains each having a light chain variable region and alight chain constant region. Further, a structure obtained byappropriately combining those structures may also be adopted. Inaddition, those structures including combined structures are eachsometimes called a fragment antibody. Such fragment antibody may be anartificially designed recombinant protein including Fv, or may be onefused with a biomolecule, such as a protein.

The Fab includes a heavy chain fragment including a heavy chain variableregion and CH1 in a heavy chain constant region, and a light chainincluding a light chain variable region and a light chain constantregion, and has a structure in which the heavy chain variable region andthe light chain variable region are associated with each other throughthe above-mentioned non-covalent intermolecular interaction, or arebonded to each other through a disulfide bond. Further, the Fab may besuch that the CH1 and the CL form a disulfide bond between the thiolgroups of cysteine residues respectively present therein.

The F(ab′)2 has a pair of the Fabs, and has a structure in which theCH1s form a disulfide bond between the thiol groups of cysteine residuesrespectively included therein.

The minibody has a pair of antibody fragments each including the scFvand CH3, and has a structure in which such antibody fragments areassociated with each other through a non-covalent intermolecularinteraction between the CH3s.

The scFv-Fc has a pair of antibody fragments each including the scFv,CH2, and CH3, and has a structure in which, as in the minibody, theantibody fragments are associated with each other through a non-covalentintermolecular interaction between the CH3s, and form a disulfide bondbetween the thiol groups of cysteine residues included in the respectiveCH3s.

A person skilled in the art can easily produce the antibody (I)including a constant region having any of such various structures aswith the antibody (I) including no constant region, by constructing anexpression vector through use of commonly used genetic engineeringmeans, and using, with such expression vector, an expression systemadopting host cells suited for antibody production. The producedantibody may be appropriately subjected to a commonly used purificationprocess so as to be obtained in a high-purity state.

The Fab may be obtained by, for example, digesting an immunoglobulin IgGwith a protease such as papain. In addition, F(ab′)₂, the F(ab′)₂ may beobtained by digesting IgG with a protease such as pepsin.

Of the above-mentioned antibodies (I) each including a constant region,a preferred structure is an immunoglobulin. The subtype of suchimmunoglobulin is not particularly limited, and examples thereof mayinclude IgA, IgD, IgE, IgG, and IgM. Of those, IgG is preferred, and forexample, in the case of IgG of mouse origin, IgG2 is preferred out ofthe four subclasses.

Of the above-mentioned antibodies (I) each including a constant region,an antibody of a more preferred embodiment is an antibody including aheavy chain having the amino acid sequence set forth in SEQ ID NO: 5and/or a light chain having the amino acid sequence set forth in SEQ IDNO: 10. The most preferred antibody is an antibody including a heavychain having the amino acid sequence set forth in SEQ ID NO: 5 and alight chain having the amino acid sequence set forth in SEQ ID NO: 10.

A mutation may be introduced into each of the above-mentioned amino acidsequences depending on the situation. It is preferred that such mutationbe not introduced into heavy-chain CDRs and light-chain CDRs. That is,the mutation is preferably introduced into a heavy-chain FR or alight-chain FR. When the antibody (I) includes a constant region, amutation may be further introduced in addition to a mutation foradjusting ADCC activity or CDC activity to be described below.

A specific number of amino acid residues at which mutations areintroduced is not particularly limited. For example, identity between anamino acid sequence before mutation introduction and an amino acidsequence after mutation introduction is about 70%, preferably about 75%,more preferably about 80%, more preferably about 85%, more preferablyabout 90%, more preferably about 95%, more preferably about 96%, morepreferably about 97%, more preferably about 98%, most preferably about99%. Such numerical value is one obtained by rounding.

The term “identity” refers to the degree of amino acid sequencesidentical to each other in two or more comparable amino acid sequences.Therefore, as the identity between given two amino acid sequencesincreases, it can be said that those sequences have not only higheridentity but also higher similarity.

The identity of amino acids may be calculated using an analytical toolthat is commercially available or available through the Internet (e.g.,software such as FASTA, BLAST, PSI-BLAST, or SSEARCH). For example, maininitial conditions to be generally used for a BLAST search are asdescribed below. That is, a value (%) for the identity between aminoacid sequences may be calculated by performing a search on AdvancedBLAST 2.1 with blastp being used as a program, the Expect value beingset to 10, all Filters being turned OFF, BLOSUM62 being used for Matrix,the Gap existence cost, Per residue gap cost, and Lambda ratio being setto 11, 1, and 0.85 (default values), respectively, and the other variousparameters being also set to default values.

The above-mentioned introduction of a mutation into an amino acidsequence refers to substitution, deletion, insertion, or the like.Specific mutation introduction is not particularly limited as long asthe mutation introduction can be achieved by adopting a commonly usedmethod. For example, in the case of the substitution, a conservativesubstitution technology may be adopted.

The term “conservative substitution technology” means a technologyinvolving substituting a certain amino acid residue with an amino acidresidue having a side chain similar thereto.

For example, substitution between amino acid residues each having abasic side chain, such as lysine, arginine, and histidine, is aconservative substitution technology. In addition, each of substitutionsbetween: amino acid residues each having an acidic side chain, such asaspartic acid and glutamic acid; amino acid residues each having anuncharged polar side chain, such as glycine, asparagine, glutamine,serine, threonine, tyrosine, and cysteine; amino acid residues eachhaving a non-polar side chain, such as alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, and tryptophan; aminoacid residues each having a β-branched side chain, such as threonine,valine, and isoleucine; and amino acid residues each having an aromaticside chain, such as tyrosine, phenylalanine, tryptophan, and histidine,is similarly a conservative substitution technology.

As another embodiment of the antibody (I), the antibody (I) may havecytotoxic activity. The cytotoxic activity refers to such activity thatthe antibody binds to cells, and as a result, causes some damage to thecells bound to the antibody.

Examples of such cytotoxic activity include ADCC activity and CDCactivity. The term “ADCC activity” is an abbreviation ofantibody-dependent cytotoxic activity (Antibody-Dependent CellularCytotoxicity), and is activity of recruiting cells having cytotoxicactivity, such as NK cells expressing a receptor specific to a constantregion of an antibody, to the vicinity of the antibody, to therebyinduce damage to cells to which the antibody binds through the action ofsuch cells and the like.

The term “CDC activity” is an abbreviation of complement-dependentcytotoxic activity (Complement-Dependent Cytotoxicity), and refers toactivity that the antibody recruits a complement to its vicinity, tothereby induce an action of causing damage to cells bound to theantibody through the action of such complement.

Here, each of the ADCC activity and the CDC activity may be adjusted byintroducing a mutation into a constant region while appropriatelyreferring to the literature, such as Lazar G A et al., Proc Natl AcadSci USA, 103: 4005-10 (2006), Shields R L et al., J Biol Chem, 276:6591-604 (2001)), Moore G L et al., J Immunol, 159:3613-21 (1997), An Zet al., MAbs, 1:572-9 (2009).

For example, when the constant region is human IgG₁, the ADCC activitymay be increased by introducing a mutation such as S239D, I332E,S239D/I332E, S239D/I332E/A330L, S298A, K334A, S298A/K334A, orS298A/E333A/K334A.

In addition, when the constant region is human IgG₁ as in the foregoing,the ADCC activity may be decreased by introducing a mutation such asV234A/G237A, or H268Q/V309L/A330S/P331S.

With regard to the CDC activity, when the constant region is human IgG₁,the activity may be increased by introducing a mutation such as S267E,H268F, S324T, S267E/H268F, S267E/S324T, H268F/S324T, orS267E/H268F/S324T.

The ADCC activity may be measured in accordance with Brunner K. T., etal.'s method (Brunner, K. T., et al., Immunology, 1968. 14:181-96). Forexample, myeloma cells are cultured in RPMI1640 medium supplemented with10% FCS, and are prepared so that the number of cells may be from0.5×10⁴ to 1.0×10⁴. An appropriate amount of Na₂ ⁵¹CrO₄ is added theretoand allowed to react therewith at 37° C. for 1 hour to label the cellswith ⁵¹Cr, and the resultant cells are washed and then used as targetcells. As effector cells, ones obtained by culturing SCID mouse bonemarrow cells for 6 days in RPMI1640 supplemented with 10% FBS, 10 ng/mlmouse GM-CSF, and 40 IU/ml human IL2, or the like may be used. To a96-well plate, an antibody to be tested or an isotype antibody thereofserving as a control is added at a final concentration from 0.05 μg/mLto 10 μg/mL, and the target cells (1.0×10⁴ cells) and the effector cells(5×10⁵ cells) are further added. The mixture is subjected to a reactionat 37° C. for 4 hours and centrifuged, and then ⁵¹Cr released into thesupernatant is measured with a γ-counter. The ADCC activity may bedetermined on the basis of the following equation.

ADCC activity={([⁵¹Cr release from target cells]-[spontaneous ⁵¹Crrelease under antibody-free state])/([maximum ⁵¹Cr release amount causedby 1% Triton X-100 addition]-[spontaneous ⁵¹Cr release underantibody-free state])}×100

The CDC activity may also be measured in accordance with Brunner K. T.,et al.'s method (Brunner, K. T., et al., Immunology, 1968. 14:181-96).For example, myeloma cells to be used as target cells are cultured inRPMI1640 medium supplemented with 10% FCS, and are prepared so that thenumber of cells may be from 0.5×10⁴ to 1.0×10⁴. An appropriate amount ofNa₂ ⁵¹CrO₄ is added thereto and allowed to react therewith at 37° C. for1 hour to label the cells with ⁵¹Cr, and the resultant cells are washedand then used as target cells. An antibody to be tested or an isotypeantibody serving as a control suspended in RPMI1640 medium supplementedwith fetal bovine serum is added to a 96-well plate at a finalconcentration of from 0.5 μg/mL to 50 μg/mL, and then the target cellsand a complement are added, followed by a reaction for 1.5 hours. Thereaction liquid is centrifuged, and ⁵¹Cr released into the supernatantis measured with a γ-counter. The CDC activity may be determined on thebasis of the following equation.

CDC activity={([⁵¹Cr release from target cells]-[spontaneous ⁵¹Crrelease under antibody-free state])/([maximum ⁵¹Cr release amount causedby 1% Triton X-100 addition]-[spontaneous ⁵¹Cr release underantibody-free state])}×100

The antibody having cytotoxic activity may be obtained by, for example,evaluating the presence or absence of cytotoxic activity through use ofthe above-mentioned method, and selecting an antibody having theactivity.

As another embodiment of the antibody (I), the antibody (I) may be amultispecific antibody. That is, the antibody (I) may have bindingcapacity with specificity to an antigen other than the region of theamino acid residue positions 20 to 109 of the human integrin β₇ (theantigen is hereinafter referred to as other antigen).

The other antigen is preferably an antigen structurally dissimilar tothe region of the amino acid residue positions 20 to 109 of the humanintegrin β₇.

A specific other antigen is not particularly limited. Examples thereofinclude CD3, CD16, C1q, and Adenovirus knob domain, and at least one ofthose antigens in appropriate combination may be appropriately adoptedas the other antigen. It is preferred that one of the antigens given asexamples above be selected as the other antigen. That is, a preferredmultispecific antibody is a bispecific antibody.

A person skilled in the art can easily produce such multispecificantibody by appropriately adopting a commonly used technology. Forexample, the multispecific antibody may be obtained in the followingmanner: hybridomas generated using antibody-producing cells, such asB-cells, obtained from an animal immunized with cells expressing apeptide fragment corresponding to the region of the amino acid residuepositions 20 to 109 of the human integrin β₇, or chimeric integrin β₇ inwhich only the region of the amino acid residue positions 20 to 109 ofthe human integrin β₇ is of human origin and the rest is of non-humanorigin, such as mouse origin, are prepared; separately, hybridomas aregenerated using antibody-producing cells, such as B-cells, obtained froman animal immunized with the above-mentioned other antigen; andscreening is performed, by a commonly used method, for new hybridomasobtained by cell fusion between the above-mentioned hybridomas (the newhybridomas are also referred to as quadromas in the case of producing abispecific antibody).

In addition to the foregoing, for example, in the case of a bispecificantibody, the bispecific antibody may be generated by a proceduredescribed in the following (1) to (4):

(1) An antibody having the structure of the above-mentioned F(ab′)₂,which uses the region of the amino acid residue positions 20 to 109 ofthe human integrin β₇ as an epitope, is generated;(2) Meanwhile, an antibody having the structure of F(ab′)₂ thatspecifically binds to the other antigen is also similarly generated;(3) The antibodies of the respective F(ab′)₂ structures obtained in (1)and (2) are treated with a reducing agent, such as DTT, and then any oneof the treated products is further treated with Ellman's reagent; and(4) The treated antibodies of the F(ab′)₂ structures obtained in (3) aremixed and allowed to react with each other.

The bispecific antibody may also be produced by a procedure described inthe following (A) to (D).

(A) An antibody using the region of the amino acid residue positions 20to 109 of the human integrin β₇ as an epitope is generated.(B) Meanwhile, an antibody that specifically binds to the other antigenis similarly generated.(C) The amino acid sequences of respective variable regions obtained in(A) and (B) and the base sequences of polynucleotides encoding thevariable regions are identified.(D) An expression vector having incorporated therein polynucleotideshaving the respective base sequences identified in (C) together with, asnecessary, polynucleotides having a base sequence for a constant domainand a linker sequence is generated, and then the expression vector isintroduced into host cells suited for antibody production, such as CHOcells.

As another embodiment of the antibody (I), the antibody (I) may havebound thereto a cytotoxin (substance having cytotoxic activity). Thecytotoxin is not particularly limited as long as the cytotoxin is asubstance that causes some damage to cells, such as the killing of cellsor the inhibition of cell growth.

Examples of such cytotoxin may include: alkylating agents, such ascyclophosphamide hydrate, iphosphamide, thiotepa, busulfan, melphalan,nimustine hydrochloride, ranimustine, dacarbazine, and temozolomide;metabolic antagonists, such as methotrexate, pemetrexed sodium hydrate,fluorouracil, doxifluridine, capecitabine, tegafur, cytarabine,gemcitabine hydrochloride, fludarabine phosphate, nelarabine,cladribine, and calcium levofolinate; antibiotic substances, such asdoxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin,epirubicin hydrochloride, idarubicin hydrochloride, aclarubicinhydrochloride, amrubicin hydrochloride, mitoxantrone hydrochloride,mitomycin C, actinomycin D, bleomycin hydrochloride, peplomycinhydrochloride, zinostatin stimalamer, and calicheamicin; microtubuleinhibitors, such as vincristine sulfate, vinblastine sulfate, vindesinesulfate, and paclitaxel; aromatase inhibitors, such as anastrozole,exemestane, letrozole, and fadrozole hydrochloride hydrate; platinatingagents, such as cisplatin, carboplatin, nedaplatin, and oxaliplatin;topoisomerase inhibitors, such as irinotecan hydrochloride hydrate,nogitecan hydrochloride, etoposide, and sobuzoxane; adrenal cortexsteroids, such as prednisolone and dexamethasone; thalidomide and aderivative thereof, specifically lenalidomide; bortezomib serving as aprotease inhibitor; and radioactive isotopes, such as 90-Ittrium.

Of those, calicheamicin, melphalan, vincristine sulfate, doxorubicinhydrochloride, prednisolone, dexamethasone, thalidomide, lenalidomide,and bortezomib are preferred, and calicheamicin having proven excellencein binding to an antibody is more preferred.

Each of those cytotoxins is commercially available, and one kind or twoor more kinds in appropriate combination may be selected from theabove-mentioned cytotoxins.

The manner of binding between the cytotoxin and the above-mentionedantibody is not particularly limited, and a person skilled in the artcan easily bind the cytotoxin to the above-mentioned antibody by, forexample, appropriately adopting a commonly used genetic engineeringtechnology or protein engineering technology. More specifically, theremay be given, for example, a method involving binding the cytotoxin to afunctional group, such as an amino group, a thiol group, a guanidylgroup, a hydroxy group, or a carboxyl group, of an amino acid residueside chain of the antibody (I) via a linker.

The antibody (I) may be a polyclonal antibody or may be a monoclonalantibody. The antibody (I) is preferably a monoclonal antibody.

The term “monoclonal” means being obtained from a substantially uniformpopulation, and the “monoclonal antibody” means an antibody obtainedfrom such population. That is, it is understood that individualantibodies included in such population are identical to each otherexcept for a naturally occurring mutation that may be present in aminute amount.

Further, with regard to a specific binding target (epitope) of anantibody, for example, in the case of the antibody (I), the epitope ispresent in the region of the amino acid residue positions 20 to 109 ofthe human integrin β₇. In this regard, in the case of a polyclonalantibody, the epitope is a plurality of sites in the region of the aminoacid residue positions 20 to 109 of the human integrin β₇, whereas inthe case of a monoclonal antibody, the epitope is a single site. Forthis reason, the monoclonal antibody exhibits high specificity, andhence is more advantageous.

The term “monoclonal” to be used as a modifier is understood to meanbeing obtained from a substantially uniform population as describedabove, and should not be understood as a modifier for specifying aproduction method for the antibody.

Other than the above-mentioned method, a person skilled in the art caneasily produce the antibody (I) by adopting a hybridoma method, arecombinant DNA method using a host cell (III) harboring apolynucleotide (II) to be described below, isolation from a phagelibrary, or the like.

For example, there may be given a method involving: immunizing an animalsuited for antibody production, such as a mouse, a rat, or a rabbit,with a peptide corresponding to the region of the amino acid residuepositions 20 to 109 of the human integrin β₇; then recovering B-cellsand subjecting the recovered B-cells to a hybridoma method; andperforming screening through use of the function exhibited by theantibody (I) described above as an indicator, to thereby produce theantibody (I).

In addition to the foregoing, there may be given a method involving:generating cells expressing chimeric integrin β₇ in which only theregion of the amino acid residue positions 20 to 109 of integrin β₇ isof human origin and the rest is of non-human origin, such as mouseorigin; immunizing an animal suited for antibody production, such as amouse, a rat, or a rabbit (preferably a mouse), with the cells; thenrecovering B-cells and subjecting the recovered B-cells to a hybridomamethod; and performing screening through use of the function exhibitedby the antibody (I) described above as an indicator, to thereby producethe antibody (I).

Examples of the function exhibited by the antibody (I) may include: anincrease in affinity for the region of the amino acid residue positions20 to 109 of the human integrin β₇ in the presence of at least part ofthe region of the amino acid residue positions 379 to 721 of the humanintegrin β₇; and an increase in the affinity through activation of thehuman integrin β7. Accordingly, the antibody (I) may be obtained by amethod shown in a screening method (X) to be described below, whichutilizes such function.

An epitope for the antibody (I) is present in the region of the aminoacid residue positions 20 to 109 of the human integrin β₇, and hence theantibody (I) is expected to exhibit cytotoxic activity or the like oncells expressing the integrin β₇ by combining one or two or more of notonly the above-mentioned ADCC activity and CDC activity, but alsoapoptosis-inducing activity, survival signal-blocking activity, and thelike on such cells. Accordingly, a composition including the antibody(I) is useful as a pharmaceutical composition (VII) as described indetail below.

In particular, the antibody (I) uses the region of the amino acidresidue positions 20 to 109 of the human integrin β₇ as an epitope, andthe affinity of the antibody (I) for the epitope is increased throughactivation of the integrin β₇. Active-form integrin β₇ is expressed inblood cells, such as plasma cells, and hence the antibody (I) is used asan active ingredient of a pharmaceutical composition for cancer thereof(e.g., blood cancer). In particular, the antibody (I) is effectivelyused as a pharmaceutical composition for a disease causing anabnormality in the above-mentioned cells (e.g., myeloma or multiplemyeloma).

(II) Polynucleotide

A polynucleotide (II) is a polynucleotide having a base sequenceencoding the amino acid sequence of the antibody (I). The term“polynucleotide” includes, for example, a single-stranded ordouble-stranded form in which a ribonucleotide, a deoxyribonucleotide,any one nucleotide thereof, or the like is appropriately modified by aknown method.

A person skilled in the art can appropriately determine the basesequence of the polynucleotide (II), for example, in silico on the basisof the amino acid sequence of the antibody (I). The kinds of codons tobe used for determining such base sequence are not limited. The basesequence is preferably determined in consideration of the codonfrequency of a host in which the polynucleotide is to be used.

A specific base sequence of the polynucleotide (II) is not particularlylimited. Correspondence between each SEQ ID NO in which an amino acidsequence identified as one embodiment of the antibody (I) is set forthand the SEQ ID NO in which a base sequence encoding such amino acidsequence is set forth is shown in Table 2 below. That is, preferred basesequences of the polynucleotide (II) are base sequences set forth in SEQID NOS: 11 to 20.

TABLE 2 Amino acid sequence Base sequence SEQ ID NO: 1 SEQ ID NO: 11 SEQID NO: 2 SEQ ID NO: 12 SEQ ID NO: 3 SEQ ID NO: 13 SEQ ID NO: 4 SEQ IDNO: 14 SEQ ID NO: 5 SEQ ID NO: 15 SEQ ID NO: 6 SEQ ID NO: 16 SEQ ID NO:7 SEQ ID NO: 17 SEQ ID NO: 8 SEQ ID NO: 18 SEQ ID NO: 9 SEQ ID NO: 19SEQ ID NO: 10 SEQ ID NO: 20

The polynucleotide (II) may adopt an embodiment of being incorporatedinto a vector. The vector is not particularly limited, and may be, forexample, a vector for cloning or a vector for expression, and itsintended use is not limited.

In addition, the vector for expression may be a vector for prokaryoticcells, such as Escherichia coli or actinomycetes, or may be a vector foreukaryotic cells, such as yeast cells, insect cells, or mammalian cells.

A base sequence encoding a signal peptide may be appropriately added tothe 5′-end side of the polynucleotide (II) (corresponding to theN-terminus side of the antibody (I)).

A specific method of using the polynucleotide (II) is not particularlylimited. For example, the polynucleotide (II) may be used for expressingthe antibody (I) by being introduced into the following host cell (III).

(III) Host Cell

A host cell (III) is a cell harboring the polynucleotide (II). The term“harbor” refers to the keeping of a state in which the polynucleotide(II) is present in the cell, and means that the cell is in a state ofnot spontaneously discharging the polynucleotide to the outside of thecell irrespective of whether the discharge is active.

The embodiment in which the host cell (III) harbors the polynucleotide(II) is not particularly limited. For example, the polynucleotide may beharbored in the form of a vector in the cell, or the polynucleotide (II)may be harbored in the form of being integrated into the genome in thecell.

A specific cell kind of the host cell (III) may be a eukaryotic cell,such as a yeast cell, an insect cell, or a mammalian cell, or may be aprokaryotic cell, such as Escherichia coli or an actinomycete, and isnot particularly limited.

(IV) Chimeric Antigen Receptor

A chimeric antigen receptor is an artificial T-cell receptor (TCR)-likeprotein, and is a protein in which an antigen recognition site expressedon the cell membrane of T-cells (corresponding to an extracellulardomain) is replaced with a desired antigen recognition site and which isconstructed so as to be able to more effectively exhibit a function,such as cytotoxic activity, of T-cells themselves.

The chimeric antigen receptor (IV) is a chimeric antigen receptor whoseepitope is identical to that of the antibody (I), and more specifically,is a protein including an antigen recognition site of the antibody (I).That is, the epitope present in the antigen recognition site included inthe chimeric antigen receptor may be the same as the one described indetail in the antibody (I).

More specifically, the chimeric antigen receptor (IV) is a protein inwhich the antigen recognition site of the antibody (I), a spacersequence, a transmembrane domain, a costimulator, and an TCRintracellular domain are arranged in the stated order from theN-terminus of the chimeric antigen receptor (IV).

The antigen recognition site of the antibody (I) to be arranged in thechimeric antigen receptor (IV) may be as described in detail in theantibody (I), and specific examples thereof may include a heavy chainvariable region and/or a light chain variable region. Of those, theantigen recognition site preferably has the structure of scFv whilehaving a heavy chain variable region and a light chain variable region.

In such scFv, for example, between the heavy chain variable region andthe light chain variable region, a spacer sequence consisting of about10 to about 25 amino acid residues may be appropriately arranged. Thenumber of amino acid residues is more preferably from about 15 to about18. Such spacer sequence may be identical to the above-mentioned spacersequence to be arranged in the chimeric antigen receptor (IV), or may bedifferent therefrom.

The spacer sequence to be arranged in the chimeric antigen receptor (IV)is not particularly limited. For example, the spacer sequence may beconsisting of about 10 to about 25 amino acid residues. The number ofamino acid residues is more preferably from about 15 to about 18.

The transmembrane domain to be arranged in the chimeric antigen receptor(IV) is not particularly limited. Specifically, a cell transmembranedomain derived from a protein, such as CD28 or 4-1BB, expressed inT-cells or the like may be adopted while being allowed to appropriatelyhave a mutation introduced thereinto.

The costimulator to be arranged in the chimeric antigen receptor (IV)may be a costimulator of T-cells or the like, and is not particularlylimited. For example, 4-1BB, OX40, CD28, or the like may be adoptedwhile being allowed to appropriately have a mutation introducedthereinto.

The TCR intracellular domain to be arranged in the chimeric antigenreceptor (IV) is not particularly limited. For example, an intracellulardomain derived from CD3, which is also called a TCR chain, or the likemay be adopted while being allowed to appropriately have a mutationintroduced thereinto. It is preferred that a mutation be introduced intoCD3 so as to include an Immunoreceptor Tyrosine-based Activation Motif(ITAM).

The chimeric antigen receptor (IV) preferably has the amino acidsequence set forth in SEQ ID NO: 21.

A mutation may be appropriately introduced into an amino acid sequenceidentifying the above-mentioned chimeric antigen receptor. In addition,a mutation may also be similarly introduced into each of theabove-mentioned transmembrane domain, costimulator, and TCRintracellular domain. A specific number of mutations to be introduced isnot particularly limited.

For example, identity between an amino acid sequence before mutationintroduction and an amino acid sequence after mutation introduction isabout 70%, preferably about 75%, more preferably about 80%, morepreferably about 85%, more preferably about 90%, more preferably about95%, more preferably about 96%, more preferably about 97%, morepreferably about 98%, most preferably about 99%. Such numerical value isone obtained by rounding.

The above-mentioned introduction of a mutation into an amino acidsequence refers to substitution, deletion, insertion, or the like.Specific mutation introduction is not particularly limited as long asthe mutation introduction can be achieved by adopting a commonly usedmethod. For example, in the case of the substitution, a conservativesubstitution technology may be adopted.

For the production of such chimeric antigen receptor, a person skilledin the art can easily produce the chimeric antigen receptor withreference to a method described in, for example, each of Non PatentLiteratures 4 to 6.

(V) Polynucleotide

A polynucleotide (V) is a polynucleotide encoding the amino acidsequence of the chimeric antigen receptor (IV) unlike the polynucleotide(II).

As in the polynucleotide (II), the base sequence of the polynucleotide(V) may be appropriately determined, for example, in silico on the basisof the amino acid sequence of the chimeric antigen receptor (IV). Thekinds of codons to be used for determining the base sequence are notlimited. The base sequence is preferably determined in consideration ofthe codon frequency of cells serving as a target for which thepolynucleotide is to be used.

A specific base sequence is not particularly limited. For example, theremay be given a polynucleotide having the base sequence set forth in SEQID NO: 22, which is determined on the basis of the amino acid sequenceof the chimeric antigen receptor (IV) having the amino acid sequence setforth in SEQ ID NO: 21. Of course, in consideration of the kinds of thecodons to be used being not limited, needless to say, the base sequenceto be determined on the basis of such amino acid sequence is not limitedto the base sequence set forth in SEQ ID NO: 22.

A base sequence encoding a signal peptide may be appropriately added tothe 5′-end side of the polynucleotide (V) (corresponding to theN-terminus side of the chimeric antigen receptor (IV)).

A specific method of using the polynucleotide (V) is not particularlylimited. For example, the polynucleotide (V) may be used for expressingthe chimeric antigen receptor (IV) by being introduced into thefollowing cell (VI).

(VI) Cell

A cell (VI) is a cell harboring the polynucleotide (V) unlike the hostcell (III). The term “harbor” may be the same as in the host cell (III).A specific kind of the cell may also be the same as in the host cell(III), but the cell (VI) preferably has cytotoxic activity. Examplesthereof may include a T-cell, an NK cell, and a K cell. Of those, akiller T-cell (sometimes referred to as cytotoxic T-cell [CTL]), whichis a kind of T-cell, is most preferred.

It is preferred that, when the polynucleotide (V), which encodes thechimeric antigen receptor, included in the cell (VI) is expressed, theantigen recognition site of the antibody (I) serving as a component ofthe chimeric antigen receptor (IV) be exposed to the outside of thecell, and the transmembrane domain, the costimulator, or the TCRintracellular domain serving as a component of the chimeric antigenreceptor (IV) be localized on a cell membrane or inside the cell.

The costimulator, or the domain to be localized on the cell membrane orinside the cell activates a signal that induces cytotoxic activity incells when the antigen recognition site of the antibody (I) binds to theregion of the amino acid residue positions 20 to 109 of the humanintegrin β₇. In addition, the affinity of the antibody (I) for theregion of the amino acid residue positions 20 to 109 of the humanintegrin β₇ is increased through activation of the human integrin β₇.Therefore, the antibody (I) attacks, or exhibits cytotoxic activity on,a cell or tissue expressing active-form integrin β₇ serving as a target.

When the cell exhibiting such function is a T-cell, the cell is referredto as chimeric antigen receptor T-cell (VI-4). Like the chimeric antigenreceptor T-cell, a cell having the possibility of exhibiting cytotoxicactivity, such as an NK cell, can also exhibit an effect similar to thatof the chimeric antigen receptor T-cell (such cell is referred to aschimeric antigen receptor NK cell) through cooperation between thebinding of the antigen recognition site to the region of the amino acidresidue positions 20 to 109 of active-form human integrin β₇, and theactivation of a signal that induces cytotoxic activity at the cellmembrane or the intracellular domain.

As described above, the cell (VI) exhibits cytotoxic activity and thelike on a cell or tissue expressing active-form integrin β₇.Accordingly, as with the antibody (I), a composition including the cell(VI) may be said to be useful as such a pharmaceutical composition (IV)as described in detail below. The active-form integrin β₇ is expressedin blood cells, such as plasma cells, and hence the cell (VI) is used asan active ingredient of a pharmaceutical composition for cancer (e.g.,blood cancer). In particular, the cell (VI) is effectively used as apharmaceutical composition for a disease causing an abnormality in theabove-mentioned cells (e.g., myeloma or multiple myeloma).

(VII) Pharmaceutical Composition

A pharmaceutical composition (VII) includes the antibody (I) or the cell(VI). The cell (VI) is preferably the chimeric antigen receptor T-cell(VI-4).

The content of the antibody (I) or the cell (VI) in the pharmaceuticalcomposition (VII) is not particularly limited. For example, in the caseof the antibody (I), the content may be from about 0.001 part by weightto about 10 parts by weight with respect to 100 parts by weight of thepharmaceutical composition. In addition, in the case of the cell (VI),the content may be from about 1 cell/mL to about 10⁴ cells/mL.

An administration method for the pharmaceutical composition (VII) is notparticularly limited. Because its active ingredient is an antibody or acell, parenteral administration or non-enteral administration ispreferred. Examples thereof include intravenous administration,intramuscular administration, and subcutaneous administration. Of those,intravenous administration is preferred.

A dosage form of the pharmaceutical composition (VII) may be preparedtogether with a pharmacologically acceptable and commonly used carrierdepending on the above-mentioned administration method. In considerationof the above-mentioned preferred administration method, the dosage formis preferably an injection.

A target disease of the pharmaceutical composition (VII) is notparticularly limited. A specific example of the target disease iscancer, preferably blood cancer, more preferably a disease causingneoplastic growth of plasma cells. The term “disease causing neoplasticgrowth of plasma cells” refers to a disease characterized by neoplasticgrowth of abnormal plasma cells and an increase in abnormal proteinsecreted therefrom. Examples of such disease may include myeloma,multiple myeloma, plasma cell leukemia, plasmacytoma, H chain disease,and systemic AL amyloidosis. In another embodiment, the target diseaseof the pharmaceutical composition (VII) may be a different hematologicmalignancy, such as malignant lymphoma or leukemia.

An administration target (subject) of the pharmaceutical composition(VII) may be a patient who has developed the above-mentioned disease, ormay be an animal having a risk of developing the above-mentioneddisease. Whether “having a risk of developing” may be determined by adiagnosis method (XI) to be described later. The animal may be, forexample, a mammal, and is preferably a human.

The dose of the pharmaceutical composition (VII) varies depending onvarious conditions of the administration target, such as the severity ofthe disease, the degree of a desired effect achieved by theadministration, body weight, sex, age, and animal species, and cannot beunconditionally determined. For example, when the active ingredient isthe antibody (I), the dose may be generally from about 1 μg/kg (bodyweight) to about 10 g/kg (body weight) per day. In addition, when theactive ingredient is the cell (VI), the dose may be generally from about10⁴ cells/kg (body weight) to about 10⁹ cells/kg (body weight).

As with its dose, the administration schedule of the pharmaceuticalcomposition (VII) also varies depending on various conditions of theadministration target, such as the severity of the disease, and cannotbe unconditionally determined. For example, the pharmaceuticalcomposition (VII) is preferably administered in the above-mentioneddaily dose at a frequency of from once a day to once a month.

(VIII) Treatment or Prevention Method for Disease

A treatment or prevention method (VIII) for a disease is a treatment orprevention method for a disease including a step of administering atherapeutically effective amount of the antibody (I) or the cell (VI) toa subject. The cell (VI) is preferably the chimeric antigen receptorT-cell (VI-4).

The subject may be the same as in the pharmaceutical composition (VII).When the subject is a patient who has developed a disease, theadministration of the therapeutically effective amount of the antibody(I) or the cell (VI) is expected to achieve a therapeutic effectthereon, and when the subject is an animal having a risk of developing adisease, the administration is expected to achieve a preventing effectthereon. As described in a diagnosis method (XI) to be described below,the preventing means keeping of a numerical value measured by a commonlyused immunological method from reaching a numerical value at which it isjudged that a disease has developed.

The disease may be the same as in the pharmaceutical composition (VII),and is exemplified by, for example, cancer. A preferred example of thecancer may be a disease causing neoplastic growth of plasma cells (e.g.,multiple myeloma).

The therapeutically effective amount may be the same as the dose of thepharmaceutical composition (VII), and a formulation of the antibody (I)or the cell (VI) may be the same as the dosage form of thepharmaceutical composition (VII). In addition, an administration methodfor the antibody (I) or the cell (VI), an administration scheduletherefor, and the like may also be as described in detail in thepharmaceutical composition (VII).

The treatment or prevention method (VIII) for a disease may encompass atreatment or prevention method for multiple myeloma including targetingactive-form human integrin β₇. An example of the targeting may beapplication of the antibody (I) or the cell (VI).

(IX) Use

A use (IX) is a use of the antibody (I) or the cell (IV), for producinga pharmaceutical composition.

The pharmaceutical composition may be the same as the pharmaceuticalcomposition (VII). The cell (IV) is preferably the chimeric antigenreceptor T-cell (VI-4).

In addition, a target disease of the pharmaceutical composition is alsothe same, and the pharmaceutical composition is used for treating, forexample, cancer, preferably blood cancer, more preferably a diseasecausing neoplastic growth of plasma cells (e.g., myeloma or multiplemyeloma).

Besides, the content of the antibody (I) or the cell (VI) serving as anactive ingredient in the pharmaceutical composition, the dosage formthereof, an administration method therefor, an administration scheduletherefor, and the like may also be the same as those described in detailin the pharmaceutical composition (VII).

(X) Screening Method

A screening method (X) is a screening method for an active ingredient ofa pharmaceutical composition for treating or preventing a diseasecausing neoplastic growth of plasma cells, including a step ofselecting, from a compound library, a candidate substance thatspecifically binds to human integrin β₇ and binds to a region of theamino acid residue positions 20 to 109 of the human integrin β₇.

The pharmaceutical composition may be the same as the pharmaceuticalcomposition (VII), and an example of an active ingredient of thepharmaceutical composition for treating or preventing cancer, preferablyblood cancer, more preferably a disease causing neoplastic growth ofplasma cells (e.g., myeloma or multiple myeloma) is the antibody (I).

The compound library is not particularly limited, and an existinglibrary may be used. The compound library is preferably an antibodylibrary, and the library preferably includes hybridomas generated usingantibody-producing cells, such as B-cells, obtained from an animalimmunized with a desired antigen.

Here, the desired antigen is not particularly limited, and ispreferably, for example, the region of the amino acid residue positions20 to 109 of the human integrin β₇. The desired antigen is morepreferably active-form human integrin β₇.

A method of selecting the candidate substance is not particularlylimited. For example, there may be adopted means involving: selecting acandidate substance that specifically binds to human integrin β₇;confirming that the candidate substance binds to a peptide fragmentcorresponding to the region of the amino acid residue positions 20 to109 of the human integrin β₇, through use of commonly used immunoassaymeans; and selecting such candidate substance.

In addition, there may be adopted means involving: selecting a candidatesubstance that specifically binds to the human integrin β₇; furtherconfirming a candidate substance that binds to cells expressing thechimeric integrin β₇ in which only the region of the amino acid residuepositions 20 to 109 of integrin β₇ is of human origin and the rest is ofnon-human origin, such as mouse origin, described in detail in theantibody (I), through use of commonly used immunoassay means; andselecting such candidate substance. Means involving selecting suchcandidate substance may also be adopted.

In addition, there may also be adopted means involving: selecting acandidate substance that specifically binds to the human integrin β₇;further treating cells expressing the chimeric integrin β₇ in which onlythe region of the amino acid residue positions 20 to 109 of integrin β₇is of human origin and the rest is of non-human origin, such as mouseorigin, described in detail in the antibody (I) with a phorbol ester, amanganese salt, or the like, and confirming a candidate substanceshowing an increased degree of binding after the treatment as comparedto that before the treatment; and selecting such candidate substance.

In addition, there may also be adopted means involving: selecting acandidate substance that specifically binds to the human integrin β₇;further generating cells expressing the chimera in which a region of theamino acid residue positions 111 to 378 of the human integrin β₇ isreplaced with one of mouse origin described in detail in the antibody(I), and a chimera in which a region of the amino acid residue positions110 to 721 of the human integrin β₇ is replaced with one of mouseorigin; confirming a candidate substance showing a higher degree of bindfor the former; and selecting such candidate substance.

Further, the screening method (X) may also include a step of selecting acandidate substance through use of the presence of cytotoxic activity asan indicator. Specific cells serving as a target on which the cytotoxicactivity is to be confirmed are not particularly limited. Examplesthereof may include blood cells expressing active-form integrin β₇ ofhuman origin having a feature in the PSI domain of the human integrin β₇described above.

Here, when the candidate substance to be screened is an antibody, thestep of selecting a candidate substance through use of the presence ofcytotoxic activity as an indicator may be a step of selecting anantibody having ADCC activity or CDC activity.

The candidate substance to be selected by the screening method (X) asdescribed above is preferably an antibody, more preferably a monoclonalantibody. The candidate substance to be selected is most preferably theantibody (I).

(XI) Diagnosis Method

A diagnosis method (XI) is a diagnosis method for cancer, and includes astep of bringing a sample collected from a subject into contact with theantibody (I).

The subject may be the same as the subject described in detail in thetreatment or prevention method (VIII) for a disease.

The sample collected from a subject may be blood or bone marrow fluid.

A specific diagnosis method is not particularly limited, but mayinvolve, for example, judging that the subject has developed, or has arisk of developing, cancer when cells that bind to the antibody (I) aredetected.

A person skilled in the art can easily determine the degree of bindingby adopting a commonly used immunoassay method. Any one of whether thesubject has developed cancer and whether the subject has a risk ofdeveloping cancer may be determined depending on the degree measuredhere.

Specific diagnosis of cancer is not particularly limited, but, forexample, the subject may be diagnosed as having developed, or having arisk of developing, blood cancer, more preferably a disease causingneoplastic growth of plasma cells (e.g., myeloma or multiple myeloma)when cells that bind to the antibody (I) are plasma cells.

(XII) Kit

A kit (XII) is a kit for diagnosis of cancer, including the antibody(I).

The cancer is not particularly limited, and may be as described indetail in the above-mentioned pharmaceutical composition (VII), and ispreferably blood cancer, more preferably a disease causing neoplasticgrowth of plasma cells (e.g., myeloma or multiple myeloma).

The kit (XII) may be appropriately accompanied by a manual. In suchmanual, the method described in detail in the above-mentioned diagnosismethod (XI) may be described to serve as a criterion for the diagnosisof cancer.

EXAMPLES

Now, Examples for describing the present invention in more detail aredescribed. Needless to say, the present invention is not limited toExamples described below.

Test Method: Flow Cytometry and Sorting

In the following Examples, flow cytometry (FACS) used for selectingcells was performed in the following manner.

Bone marrow mononuclear cells collected from an ilium of a myelomapatient who had given informed consent were suspended in an ACK solution(150 mM NH₄Cl and 10 mM KHCO₃), and the whole was left at rest at 4° C.for 3 minutes to remove red blood cells. The bone marrow mononuclearcells after the removal were washed with PBS supplemented with 2% fetalbovine serum, and then, in order to prevent the binding of anon-specific antibody, blocking was performed at 4° C. for 20 minutes inPBS containing 10% human AB serum.

After that, each antibody (see below) labeled with a fluorescent dye wasadded thereto to perform staining at 4° C. for 30 minutes. After that,the cells were washed with PBS, and then suspended in PBS containing 1μg/ml propidium iodide (PI), followed by FACS analysis. The analysis ofthe cells and cell sorting were performed using FACS Aria Cell Sorter(manufactured by Becton Dickinson Immunocytometry Systems).

For the staining of the cells, the following monoclonal antibodies wereappropriately selected and used.

-   -   APC-conjugated anti-human CD34 antibody (manufactured by BD        Pharmingen)    -   PE-Cy7-conjugated anti-human CD34 antibody (manufactured by BD        Pharmingen)    -   APC/Cy7-conjugated anti-human CD19 antibody (manufactured by        Biolegend)    -   FITC-conjugated anti-human CD38 antibody (manufactured by        eBioscience)    -   APC-conjugated anti-human CD138 antibody (manufactured by        Biolegend)    -   PE/Cy7-conjugated anti-human CD3 antibody (manufactured by        Biolegend)    -   FITC-conjugated anti-human CD14 antibody (manufactured by BD        Pharmingen)    -   PE/Cy7-conjugated anti-human CD45 antibody (manufactured by        Biolegend)

Example 1

Generation of Monoclonal Antibody Library that binds to Myeloma CellLine and does not bind to Healthy Person Peripheral Blood

In antibody therapy against multiple myeloma, it is important to use anantibody that binds to myeloma cells but does not bind to normal bloodcells. In view of this, such antibody was identified by the followingmethod. First, 10,000 clones or more of monoclonal antibodies that boundto various myeloma cell lines were generated using the followingtechnique.

Balb/c mice were immunized at the footpad twice a week for from 2 weeksto 3 weeks through use of six kinds of human myeloma cell lines (MM.1scells, RPMI8226 cells, INA6 cells, U266 cells, OPM2 cells, and KMS12BMcells) as antigens. After that, a lymph node below the knee was removed,and a cell suspension was generated and subjected to cell fusion with aSP2/0 mouse myeloma cell line to generate hybridomas. The cell fusionwas performed using a method using polyethylene glycol (PEG method).After that, the cells were cultured inhypoxanthine-aminopterin-thymidine medium (HAT medium) to selecthybridomas (>10,000 clones).

Finally, through use of culture supernatants of the hybridomas, asupernatant including antibodies that bound to the myeloma cell lineused for the immunization and did not bind to healthy person peripheralblood-derived mononuclear cells was selected using FACS. Antibodycandidates specific to myeloma cells obtained as a result of theforegoing were about 200 clones, and the hybridomas expressing thoseantibody candidates were grown and then cryopreserved.

Example 2

Identification of Antibody that specifically binds to Myeloma Cells inHuman Multiple Myeloma Patient Bone Marrow

About 200 clones of candidate antibodies obtained in Example 1 describedabove were used to stain myeloma patient-derived bone marrow cells,followed by analysis using FACS.

Each candidate antibody was added to multiple myeloma patient-derivedbone marrow cells, and the cells were incubated at 4° C. for 30 minutesand then washed. A PE-conjugated anti-mouse IgG antibody was added as asecondary antibody, and the cells were further incubated at 4° C. for 30minutes. After washing, finally, the cells were stained using anAPC-conjugated anti-human CD138 antibody, FITC-conjugated anti-humanCD38, or PE/Cy7-conjugated anti-human CD45. As a negative control, asample having added thereto an isotype control in place of the candidateantibody was prepared as the same time.

Those cells were analyzed using FACS to select an antibody that bound toCD45⁻CD38⁺⁺CD138⁺ myeloma plasma cells and CD45⁻CD38++CD138⁻ myelomaprogenitor cells, but did not bind to CD45⁺ blood cells.

As a result, an MMG49 antibody was identified as an antibody satisfyingthe above-mentioned condition (FIG. 1 and FIG. 2). In each histogram inthe figures, the Y-axis represents the number of cells, and the X-axisrepresents the binding strength of the MMG49 antibody.

Example 3

Identification of Antigen Protein to which MMG49 Antibody binds

An antigen protein to which the MMG49 antibody bound was identified byan expression cloning method.

First, a cDNA library was generated from MM.1s cells, to which the MMG49antibody was known to bind, using a superscript choice system for cDNAsynthesis (Invitrogen), and was inserted into a pMXs retrovirus vector(donated by Professor Toshio Kitamura at the Institute of MedicalScience of the University of Tokyo) using a BstXI adaptor (Invitrogen).The thus generated cDNA library was introduced into plat-E cells(donated by Professor Toshio Kitamura), and BaF3 cells were infectedwith the resultant retrovirus. Thus, BaF3 cells expressing anMM.1s-derived cDNA library were obtained.

Next, the cells were repeatedly concentrated by staining the cells withthe MMG49 antibody and sorting positive cells by FACS (FIG. 3). Afterthe third sorting, most cells were cells that bound to the MMG49antibody. Then, the retrovirus insert carried by those cells wasamplified by PCR, and then sequenced to identify its base sequence. As aresult, it was revealed that the insert carried by the cells was ITGB7.

Example 4

Confirmation that Antigen to which MMG49 Antibody binds isITGB7-expressed Protein by Generation of ITGB7-deficient Myeloma Cells

An ITGB7-deficient U266 myeloma cell line was generated using aCrisp-cas9 system.

First, a vector was generated by inserting a double-stranded DNAsequence serving as an ITGB7-specific target sequence into a PX330(addgene) vector. The vector was introduced together with a linearhygromycin-resistance gene expression vector (Clontech) serving as avector for drug selection into U266 cells through use of Nucleofector(trademark) II (Lonza). After that, for clones that had grown in amedium supplemented with hygromycin, the expression of ITGB7 was stainedusing an FIB27 antibody (anti-integrin β₇ antibody; Biolegend), followedby analysis by FACS, to thereby identify ITGB7-deficient cells.

Next, the resultant ITGB7-deficient cells were stained using the MMG49antibody and analyzed by FACS. As a result, it was found that the MMG49antibody bound to wild-type U266 cells, whereas the binding of the MMG49antibody had completely disappeared in the ITGB7-deficient strain (FIG.4). This shows that MMG49 is bound to only the ITGB7-expressed protein(integrin β₇).

Next, immunoprecipitation from a lysate of MM1s myeloma cells wasperformed using the MMG49 antibody, followed by SDS-PAGE. Subsequently,WB was performed using an anti-integrin β₇ antibody (Miltenyi). As aresult, integrin β₇ was detected in the product immunoprecipitated withthe MMG49 antibody (FIG. 5). This shows that the MMG49 antibody is boundto the integrin β7.

Example 5 Measurement of Binding Pattern of MMG49 Antibody in CellFractions of Healthy Person Peripheral Blood and Myeloma Patient BoneMarrow

Through use of a commercially available anti-integrin β₇ antibody (FIB27antibody; Biolegend) and the MMG49 antibody, binding to various cellfractions in healthy person peripheral blood and bone marrow cells wasmeasured.

Red blood cells were removed from healthy person-derived peripheralblood cells using HES40, and then an Fc receptor blocking reagent(Miltenyi) was added to block non-specific antibody binding. After that,the MMG49 antibody or the FIB27 antibody, or mouse IgG2a serving as anisotype control was added, and the cells were incubated at 4° C. for 30minutes and then washed. A PE-conjugated anti-mouse IgG antibody wasadded as a secondary antibody, and the cells were further incubated at4° C. for 30 minutes.

The resultant cells were washed, and then, finally, stained using anAPC/Cy7-conjugated anti-human CD19 antibody, an FITC-conjugatedanti-human CD14 antibody, or a PE/Cy7-conjugated anti-human CD3antibody. The cells after the staining were analyzed using FACS, tothereby measure the binding of the MMG49 antibody and the FIB27 antibodyin each fraction (FIG. 6).

In addition, 100 μl of PBS (containing EDTA) supplemented with 1 μl ofperipheral blood of a healthy person was similarly stained using theMMG49 antibody or the FIB27 antibody, and finally stained using aPacific blue-conjugated anti-human CD235 antibody (BD Pharmingen) or anFITC-conjugated anti-human CD41 antibody (BD Pharmingen), and thus thepresence or absence of the binding of each antibody to CD235⁺ red bloodcells and platelets was also similarly investigated by FACS analysis(FIG. 6). The results of the foregoing show that the FIB27 antibodystrongly binds to many lymphoid cells, whereas the binding of the MMG49antibody to the above-mentioned normal blood cells is extremely weak.

Further, in order to elucidate whether the binding of the MMG49 antibodyto each normal cell fraction except for myeloma cells was absent in bonemarrow, bone marrow cells of a myeloma patient were also similarlystained using the MMG49 antibody, and finally stained using anAPC-conjugated anti-human CD34 antibody (manufactured by BD Pharmingen),Alexa647-conjugated human CD3 (manufactured by BD Pharmingen), aCy7APC-conjugated anti-CD19 human antibody (manufactured by BDPharmingen), a PE-Cy7-conjugated anti-CD38 human antibody (manufacturedby BD Pharmingen), or an FITC-conjugated anti-CD14 human antibody(manufactured by BD Pharmingen). Those myeloma patient-derived bonemarrow cells after the staining were analyzed using FACS, and thus thebinding of the MMG49 antibody in each fraction was measured (FIG. 7).The results of the foregoing show that the MMG49 antibody strongly bindsto myeloma cells, but hardly binds to all normal blood cells includinghematopoietic stem cell and progenitor cell fractions.

Example 6 Analysis of Binding of MMG49 to Various Cell Lines

The binding of the MMG49 antibody and the FIB27 antibody in various celllines (MM1s cells, U266 cells, RPMI8226 cells, and JJN3 cells) wasanalyzed using FACS. A staining method is the same as in the case ofperipheral blood or the like described above in Example 5.

Integrin β₇ is known to form a heterodimer with integrin α₄ or integrinα_(E) and be expressed on a cell surface. Therefore, the expressionthereof was also analyzed at the same time using FACS, with anAlexa647-cojugated anti-human CD49d antibody (Biolegend) and anAPC-conjugated anti-human CD103 antibody (Biolegend). CD103 representsthe integrin α_(E), and CD49d represents the integrin α₄. In the samemanner as in Example 5 described above, the expression levels of theintegrin α_(E) and the integrin α₄ in healthy person-derived peripheralblood were also investigated (FIG. 8).

As a result, ITGA4 was expressed in most of the myeloma cell lines, andITGAE was expressed in none of the cell lines. The FIB27 antibody wasbound to all myeloma cell lines, but the binding of the MMG49 antibodydid not coincide with the expression level of the FIB27 antibody.Further, the binding of the MMG49 antibody or the FIB27 antibody toITGA4-deficient U266 cells generated using a Crisp-cas9 system wasinvestigated by FACS, and as a result, it was found that the binding ofboth the antibodies to U266 cells had disappeared due to ITGA4deficiency. That is, it was found that both the MMG49 antibody and theFIB27 antibody recognized β₇ integrin expressed as α₄β₇ integrin.

Example 7

Analysis of Correlation between Activation of Integrin and Binding ofMMG49 Antibody

In consideration of the unique binding manner of the MMG49 antibodydescribed above, it was supposed that the MMG49 antibody recognizedintegrin β₇ that had been changed in structure through activation.

In view of the foregoing, K562 cells caused to forcibly express α₄β₇ andhuman normal peripheral blood CD4T-cells concentrated using a CD4 T-cellenrichment kit (BD pharmingen) were washed with 5 mM EDTA/HBS, and thenincubated in 1 mM Ca²⁺/1 mM Mg²⁺/HBS (buffer for low activity) or 2 mMMn²⁺/HBS (activating buffer) together with the MMG49 antibody or theFIB27 antibody at room temperature for 30 minutes, followed by washing.A PE-conjugated anti-mouse IgG antibody was added as a secondaryantibody, and the cells were further incubated at room temperature for30 minutes. The resultant cells were analyzed using FACS, to therebymeasure the binding of the MMG49 antibody and the FIB27 antibody incells in which the integrin α₄β₇ had been activated.

As a result, an enhancement in binding of the MMG49 antibody in thepresence of Mn²⁺ was observed (FIG. 10). Meanwhile, no similar changewas observed for the FIB27 antibody. This suggests the possibility thatthe MMG49 antibody is an antibody specific to activated conformation ofintegrin β₇.

Example 8

Identification of Epitope essential for Recognition by MMG49 Antibody

In order to identify an epitope recognized by the MMG49 antibody,vectors for expressing eight kinds of human/mouse chimeric integrin β₇proteins as illustrated in FIG. 11 were generated using an overlappingPCR method.

Each expression vector was introduced into 293T-cells by a lipofectionmethod, and 48 hours after that, the presence or absence of the bindingof the MMG49 antibody was analyzed. The cells were suspended in PBSsupplemented with 1% fetal bovine serum, the MMG49 antibody was added,and then the whole was left at rest at room temperature for 30 minutes.After washing, an Alexa488-anti-mouse IgG antibody was added, and thewhole was left at rest at room temperature for 30 minutes, followed byanalysis by FACS.

As a result, it was revealed that the MMG49 antibody strongly bound to achimeric integrin β₇ protein (#4960) in which a region of the amino acidresidue positions 110 to 721 was of mouse origin and the rest (region ofthe amino acid residue positions 20 to 109 and region of the amino acidresidue positions 722 to 798) had sequences of human origin in almostthe same manner as in the case of a chimeric integrin β₇ proteinentirely of human origin (#4927) (FIG. 11 to FIG. 13).

In view of the fact that the region of the amino acid residue positions722 to 798 includes a transmembrane domain (TM) and an intracellulardomain (cytoplasmic), and that a region of the amino acid residuepositions 1 to 19 is a signal peptide, it was shown that an epitopeessential for the binding of the MMG49 antibody was present in theregion of the amino acid residue positions 20 to 109 including a PSIdomain.

In addition, it was revealed that the MMG49 antibody had a slightlyincreased avidity for a chimeric integrin β₇ protein (#4961) in which aregion of the amino acid residue positions 110 to 378 was of mouseorigin, and in which a region of the amino acid residue positions 20 to109 and a region of amino acid residue positions 379 to 798 were ofhuman origin as compared to the chimeric integrin β₇ protein (#4960),and the increased avidity was at exactly the same binding level as inthe case of the chimeric integrin β₇ protein entirely of human origin(#4927).

Further, it was also revealed that the MMG49 antibody had slightlyincreased binding capacity for a chimeric integrin β₇ protein (#4944) inwhich the above-mentioned region of the amino acid residue positions 20to 109, which had been shown to include the epitope of the MMG49antibody, and a region of the amino acid residue positions 1 to 378including a region corresponding to a signal peptide of the amino acidresidue positions 1 to 19 were of mouse origin, and in which a region ofthe amino acid residue positions 379 to 798 was of human origin, and achimeric integrin β₇ protein (#4945) in which a region of the amino acidresidue positions 1 to 416 was of human origin and a region of the aminoacid residue positions 417 to 798 was of human origin as compared to achimeric integrin β₇ protein (#4946) in which a region of the amino acidresidue positions 1 to 563 was of mouse origin and a region of the aminoacid residue positions 564 to 798 was of human origin, and a chimericintegrin β₇ protein (#4947) in which a region of the amino acid residuepositions 1 to 721 was of mouse origin and a region of the amino acidresidue positions 722 to 798 was of human origin.

In view of the above-mentioned experimental results, it was alsorevealed that the specific avidity, that is, affinity of the MMG49antibody for the region of the amino acid residue positions 20 to 109 ofthe integrin β₇ was increased by the region of the amino acid residuepositions 379 to 721 of human integrin β₇, that is, in the presence ofthe region of the amino acid residue positions 379 to 721 of humanintegrin β₇.

Example 9 Determination of Base Sequence of Antibody Molecule VariableRegions of MMG49 Antibody

The subclass of the MMG49 antibody was confirmed using an Isotyping kit(Roche), and was confirmed to be IgG2a subclass. Further, the basesequences and amino acid sequences of variable regions of the MMG49antibody were determined.

A sequence determination method was performed using a Smarter RACE cDNAamplification kit (Clontech). That is, cDNAs generated from mRNAsderived from hybridomas MMG49 producing the MMG49 antibody were used astemplates to amplify cDNA fragments of H-chain and κ-chain variableregions by a PCR reaction, and their base sequences were read. The readamino acid sequence and base sequence, and hypervariable regions (CDR1to CDR3) of the H-chain variable region are shown in Tables 3 and 4below.

The read amino acid sequence and base sequence, and hypervariableregions (CDR1 to CDR3) of the L-chain (κ-chain) variable region are alsoshown in Tables 3 and 4 below.

In order to confirm the specificity of the isolated variable regionsequences of the MMG49 antibody, variable region sequence cDNAs werebound to a human IgG4 constant domain and human IgL κ-chain constantdomain sequence to generate a chimerized antibody. Specifically, eachvariable region sequence was inserted into pFuse-CH-Ig-hG4 orpFuse-CL-Ig-hk (invivogen) using In-Fusion cloning kit (Takara). Afterthat, the resultant was introduced into FreeStyle CHO-S cells(Invitrogen), and a chimerized antibody secreted into the culturesupernatant thereof was recovered. Next, MM1s cells to which the MMG49antibody bound and KMS12BM cells to which the MMG49 antibody did notbind were incubated in a buffer supplemented with MMG49-hIgG4, and werewashed. After that, biotinylated anti-human IgG (Rockland) was added asa secondary antibody, and the cells were washed again, and then stainedby adding streptavidin-PE (Biolegend), followed by FACS analysis. As aresult, MMG49-hIgG4 showed a staining pattern similar to that of theoriginal MMG49 antibody, suggesting that the obtained variable regionsequences were correct (FIG. 14).

<Table 3: Amino acid sequences of M4G49> Heavy CDR1 (SEQ ID NO: 1)GYTFSSYW chain CDR2 (SEQ ID NO: 2) MLPGSGSS CDR3 (SEQ ID NO: 3)ARGDGNYWYFDV Variable region MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKI(SEQ ID NO: 4) SCKASGYTFSSYWIEWVKQRPGHGLEWIGEMLPGSGSSNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCAR GDGNYWYFDVWGAG LightCDR1 (SEQ ID NO: 6) SSVGY chain CDR2 (SEQ ID NO: 7) ATSCDR3 (SEQ ID NO: 8) QQWSSDPPT Variable regionMDFQVQIFSFLLISASVIMSRGQIVISQSPAILSASPGE (SEQ ID NO: 9)KVTMTCRASSSVGYMHWFQQKPGSSPKPWIYATSNLASGVPARFSGSESGTSYSLTISRVEAEDAATYYCQQWSSDPP TFGGGTKLEIK

<Table 4: Base sequences of MMG49> Heavy CDR1 (SEQ ID NO: 11)GGCTACACATTCAGTAGCTACTGG chain CDR2 (SEQ ID NO: 12)ATGTTACCTGGAAGTGGTAGTTCT CDR3 (SEQ ID NO: 13)GCAAGGGGGGATGGTAACTACTGGTACTTCGATGTC Variable regionATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCA (SEQ ID NO: 14)GTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTTCTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGGGATGGT AACTACTGGTACTTCGATGTCTGGGGCGCAGGGLight CDR1 (SEQ ID NO: 16) TCAAGTGTAGGTTAC chain CDR2 (SEQ ID NO: 17)GCCACATCC CDR3 (SEQ ID NO: 18) CAGCAGTGGAGTAGTGACCCACCGACGVariable region ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTA (SEQ ID NO: 19)ATCAGTGCTTCAGTCATAATGTCCAGAGGACAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAGGTTACATGCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGAGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCACCGACGTTCGGT GGAGGCACCAAGCTGGAAATCAAA

Example 10

Generation of Chimeric Antigen Receptor T-cells using Antibody MoleculeVariable Regions of MMG49 Antibody

Chimeric antigen receptor T-cells (hereinafter referred to as MMG49antibody-derived chimeric antigen receptor T-cells) were generated usingthe MMG49 antibody molecule variable region sequences by the followingprocedure with reference to Non Patent Literatures 2 to 4 and the like.

(1) Cloning of CD28 and CD3z:

RNAs were collected from Jurkat cells through use of Trizol(Invitrogen), and cDNAs were generated using a Superscript III cDNAsynthesis kit (Invitrogen). Then, the cDNAs were used as templates toamplify cDNAs of CD28 and CD3z by PCR, each of which was cloned using aTA cloning kit (Invitrogen), and their base sequences were confirmed bysequencing.

(2) Binding of Four Fragments, i.e., MMG49 antibody-derived VL/VH, andCD28/CD3z:

Through use of an overlapping PCR method, respective gene fragments ofthe MMG49 antibody-derived VL region and VH region, and the CD28 andCD3z cloned above were bound to each other to generate chimeric cDNA. Aprocedure therefor and primers used are illustrated in FIG. 15. The basesequences of the primers used are shown in Table 5 below.

TABLE 5 No. Primer name Base sequence 23 49_car_vk-s5gaattccaccatggattttcaagtgcagatt 24 49_car_vk-as6gccggaaccgctagtggagccccgtttgatttccagcttggt 25 593_car_vk as4gctgccttctccgctgccaggtttgccggaaccgctagtggagcc 26 49_car_vh-s5aaacctggcagcggagaaggcagccaggttcagctgcagcagtc 27 49_car_vh-as6tgaggagacggtgaccgtgg 28 49_VEVE28as8atacataacttcaattgcggccgctgaggagacggtgaccgtgg 29 49carinfus1ctaggcgccggaattccaccatggattttc 30 tcrzcarinfuas4aatgtcgacctcgagtggctgttagcgag

The bound chimeric cDNA was cloned using a Zeroblunt PCR cloning kit(Invitrogen), and then sequenced to confirm its base sequence. Inaddition, an amino acid sequence (SEQ ID NO: 21) confirmed on the basisof the confirmed base sequence and the base sequence (SEQ ID NO: 22) areshown in the sequence listing. The amino acid sequence set forth in SEQID NO: 21 is one obtained by conversion into an amino acid sequence fromthe start codon (atg) immediately following the Kozak sequence(gaattccacc) shown above in SEQ ID NO: 23, which was excluded.

(3) Insertion into Expression Vector:

Subsequently, the chimeric cDNA bound in (2) was cleaved with tworestriction enzymes EcoRI/SalI, and inserted into an MSCV-ires-GFPvector.

The MMG49 antibody-derived chimeric antigen receptor cDNA retrovirusvector generated by the foregoing was introduced into 293T cellstogether with a gag/pol and VSV-G envelope expression vector through useof lipofectamine 2000 (invitrogen), to thereby generate a retrovirus.After 48 hours from the gene introduction, a supernatant was recoveredand used as a virus solution.

(4) Introduction into T-cells:

Subsequently, cDNA of an MMG49 antibody-derived chimeric antigenreceptor was introduced into human T-cells as described below.

First, human peripheral blood mononuclear cells were added to a 48-wellplate coated with an anti-CD3 antibody (eBioscience) and cultured for 72hours. X-VIVO15 (Lonza) supplemented with 10% human AB serum and IL-2(175 IU/L) was used as a culture medium, and the peripheral bloodmononuclear cells were stimulated. After that, the virus solutiongenerated above was added to a 48-well plate coated with Retronectin(Takara), and the virus was adsorbed to Retronectin by centrifugation at1,700×g for 120 minutes. After that, the peripheral blood mononuclearcells (including T-cells) after the stimulation were added, and the genewas introduced thereinto. After that, culture was continued in theabove-mentioned medium to amplify MMG49 antibody-derived chimericantigen receptor T-cells, which were used in the followinginvestigation. The T-cells caused to express a CAR construct usingvariable regions of the MMG49 antibody were stained using aPE-anti-human F(ab′)₂ antibody (Jackson Laboratory), and as a result,the expression of human F(ab′)₂ was detected in proportion to theexpression of GFP indicating the introduction of the construct (FIG.16). That is, it was confirmed that the introduced CAR was expressed ona cell surface.

Example 11

Analysis of Recognition of ITGB7-expressing Tumor Cells by MMG49Antibody-derived Chimeric Antigen Receptor T-cells and CytotoxicActivity thereof

The MMG49 antibody-derived chimeric antigen receptor T-cells generatedby the above-mentioned method or control T-cells having introducedtherein only GFP were cocultured with K562 cells expressing no integrinβ₇ or K562 cells caused to forcibly express integrin α₄β₇, and theamount of a produced cytokine was quantitatively determined.Specifically, 1×10⁵ each of the T-cells and the target cells were addedto a 96-well plate. After 24 hours, a supernatant was recovered, and theamount of production of IFN-γ was measured by ELISA. The measurement wasperformed using a Quantikine kit (R&D). As a result, only in thecoculture of the K562 cells caused to forcibly express integrin α₄β₇with the MMG49 antibody-derived chimeric antigen receptor T-cells,higher production of IFN-γ and IL2 than in the control (T-cells obtainedby similarly culturing stimulated peripheral blood mononuclear cellshaving introduced therein a GFP expression vector) was observed (FIG.17).

Next, the MMG49 antibody-derived chimeric antigen receptor T-cells orcontrol T-cells having introduced therein only GFP were cocultured withmyeloma cell lines to which the MMG49 antibody bound (MM.1s cells,RPMI8226 cells, and JJN3 cells) or cells to which the MMG49 antibody didnot bind (KMS12BM, Molt4, and Raji cells), and the amount of a producedcytokine was similarly quantitatively determined. As a result, only whenthe MM.1s, RPMI8226 cells, and JJN3 cells, which were cells to which theMMG49 antibody bound, were cocultured with the MMG49 antibody-derivedchimeric antigen receptor T-cells, higher production of IFN-γ and IL2than in the control (T-cells obtained by similarly culturing stimulatedperipheral blood mononuclear cells having introduced therein a GFPexpression vector) was observed (FIG. 18 and FIG. 19). The results showthat the MMG49 antibody-derived chimeric antigen receptor T-cells areactivated by recognizing an antigen recognized by the MMG49 antibody(sometimes referred to as MMG49 antigen).

Further, whether the MMG49 antibody-derived chimeric antigen receptorT-cells damaged a myeloma cell line was investigated by ⁵¹Crcytotoxicity assay. First, K562 cells expressing no integrin β₇ or K562cells caused to forcibly express integrin α₄β₇ to be used as targetcells were cultured in RPMI1640 medium supplemented with 10% FCS, andwere prepared so that the number of cells was from 0.5×10⁴ to 1.0×10⁴.

An appropriate amount of Na₂ ⁵¹CrO₄ was added thereto and allowed toreact therewith at 37° C. for 2 hours to label the cells with ⁵¹Cr, andthe resultant cells were washed and then used as target cells. Thetarget cells were mixed with MMG49 antibody-derived chimeric antigenreceptor T-cells suspended in RPMI1640 medium supplemented with fetalbovine serum, and the cells were cocultured for 4 hours.

After that, ⁵¹Cr released into the supernatant was measured with aγ-counter. A cell damage percentage (%) was determined on the basis ofthe following expression (1).

(A−B)/(C−D)×100  (1)

A: Amount of ⁵¹Cr released from cells used in experimentB: Spontaneous ⁵¹Cr release amount under antibody-free stateC: Maximum ⁵¹Cr release amount with addition of 1% Triton X-100D: Spontaneous ⁵¹Cr release amount under antibody-free state.

As a result, in the K562 cells caused to forcibly express integrin α₄β₇to which the MMG49 antibody bound, higher cell damage caused by theMMG49 antibody-derived chimeric antigen receptor T-cells than in theT-cells expressing only GFP serving as a control was observed (FIG. 20).

Next, the MMG49 antibody-derived chimeric antigen receptor T-cells orcontrol T-cells having introduced therein only GFP were cocultured withmyeloma cell lines to which the MMG49 antibody bound (MM1s cells,RPMI8226 cells, and JJN3 cells) or cells to which the MMG49 antibody didnot bind (KMS12BM, Molt4, and Raji cells), and a similar investigationwas performed. As a result, only in the K562 cells caused to forciblyexpress integrin α₄β₇ to which the MMG49 antibody bound, higher celldamage caused by the MMG49 antibody-derived chimeric antigen receptorT-cells than in the T-cells expressing only GFP serving as a control wasobserved (FIG. 21).

The above-mentioned results show that the MMG49 antibody-derivedchimeric antigen receptor T-cells can specifically damage cellsexpressing an antigen to be recognized by the MMG49 antibody.

Example 12

Analysis of Myeloma Cell-eliminating Ability exhibited by MMG49Antibody-derived Chimeric Antigen Receptor T-cells in vivo

A therapeutic effect on multiple myeloma was investigated in vivo usingthe MMG49 antibody-derived chimeric antigen receptor T-cells.

Myeloma cell line MM1s cells (4×10⁵ cells) were transplanted into thebone marrow of NOG mice subjected to radiation exposure at 2.4 Gy. After5 days, the mice were grouped into an MMG49 antibody-derived chimericantigen receptor T-cell-administered group and a controlT-cell-administered group, and each group was intravenously administeredwith 5×10⁶ cells per mouse. After 7 days from the administration, thebone marrow was analyzed. As a result, marked growth of myeloma cellswas clearly observed in all mice of the control T-cell-administeredgroup, whereas the tumor had almost completely disappeared in the MMG49antibody-derived chimeric antigen receptor I-cell-administered group.The results show that the administration of the MMG49 antibody-derivedchimeric antigen receptor T-cells has an ability to eliminate a tumorexpressing the MMG49 antigen even in vivo (FIG. 22).

Further, a therapeutic effect on multiple myeloma was investigated invivo using a myeloma-systemically seeded model.

Myeloma cell line MM1s cells (5×10⁶ cells) having introduced thereinluciferase gene were intravenously transplanted into NOG mice subjectedto radiation exposure at 2.4 Gy. After 5 days from the transplantation,the degree of engraftment of the tumor cells was measured using an IVISimaging system (PerkinElmer). After that, the mice were grouped into anMMG49 antibody-derived chimeric antigen receptor T-cell-administeredgroup and a control T-cell-administered group, and each group wasintravenously administered with 3×10⁶ cells per mouse 5 days and 7 daysafter the transplantation. After 7 days from the second T-celladministration, a tumor volume was measured again using the IVIS imagingsystem. As a result, marked growth of myeloma cells was clearly observedin all mice of the control T-cell-administered group, whereas the tumorhad almost completely disappeared in the MMG49 antibody-derived chimericantigen receptor T-cell-administered group (FIG. 23). The results showthat the administration of the MMG49 antibody-derived chimeric antigenreceptor T-cells has an ability to eliminate a tumor expressing theMMG49 antigen even in vivo.

Example 13

For the epitope of the MMG49 antibody, an experiment for investigating,in more detail, the results of the investigation in Example 8 wasperformed. Vectors for expressing three kinds of human/mouse chimericintegrin β₇ protein as illustrated in FIG. 25 were generated, and eachof the expression vectors was introduced into 293T cells by alipofection method. After 48 hours, the presence or absence of thebinding of the MMG49 antibody was analyzed by FACS.

As a result, it was revealed that the MMG49 antibody strongly bound to achimeric integrin β₇ protein (ch5.1 in FIG. 25) in which regions of theamino acid residue positions of 1 to 32 and positions 91 to 798 ofintegrin β₇ protein were of mouse origin, and the rest (region of theamino acid residue positions 33 to 90) had a sequence of human origin inalmost the same manner as in the case of the integrin β₇ proteinentirely of human origin (#4927 in FIG. 11) (FIG. 25).

Thus, it was strongly suggested that the epitope of the MMG49 antibodywas included in the amino acid residues at positions 33 to 90 of thehuman integrin β₇ protein.

Example 14

Vectors expressing the human integrin β₇, the mouse integrin β₇, andvarious variants obtained by mutating only one or two amino acids of thehuman integrin β₇ into an amino acid sequence of mouse origin(R35E/N36D, H38D, M41L/L42Q, and A48V) were introduced into 293T cellsby a lipofection method, and an experiment was performed in the samemanner as in Example 8 thereafter. As a result, as shown in FIG. 26, itwas revealed that only the A48V variant had a remarkably reduced avidityfor the MMG49 antibody as compared to the human integrin β₇, and had anumerical value close to that of the mouse integrin β₇. The resultsrevealed that the amino acid residue at position 48 of the humanintegrin β₇ was strongly related to the epitope of the MMG49 antibody,or included in the epitope of the MMG49 antibody.

Base sequences and amino acid sequences described herein are shownbelow.

1-15. (canceled)
 16. A chimeric antigen receptor, comprising ananti-integrin β₇ scFv, wherein the anti-integrin β₇ scFv comprises: aheavy chain variable region comprising an amino acid sequence having atleast 80% identity with the amino acid sequence of SEQ ID NO: 4, and alight chain variable region comprising an amino acid sequence having atleast 90% identity with the amino acid sequence of SEQ ID NO:
 9. 17. Thechimeric antigen receptor of claim 16, wherein the heavy chain variableregion comprises the amino acid sequence of SEQ ID NO: 1, the amino acidsequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3,and wherein the light chain variable region comprises the amino acidsequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, andthe amino acid sequence of SEQ ID NO:
 8. 18. The chimeric antigenreceptor of claim 16, which binds to a region of amino acid residuepositions 20 to 90 of an anti-integrin β₇.
 19. The chimeric antigenreceptor of claim 16, which is multispecific.
 20. A cell comprising thechimeric antigen receptor of claim
 16. 21. The cell according to claim20, which is a chimeric antigen receptor T-cell or NK-cell.
 22. Apharmaceutical composition comprising the cell according to claim 20.23. A method of treating multiple myeloma comprising administering thecell of claim 20 to a patient in need thereof.
 24. A pharmaceuticalcomposition comprising a dose from 10⁴ cells/kg to 10⁹ cells/kg of thecell according to claim 20.